Anti-huLRRC15 antibody drug conjugates and methods for their use

ABSTRACT

The present disclosure provides antibodies, antibody binding fragments, and antibody drug conjugates that bind human LRRC15, their methods of making, and their uses to treat patients having cancer.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application No. 62/261,092, filed Nov. 30, 2015, and U.S.provisional application No. 62/417,480, filed Nov. 4, 2016, the contentsof both of which are incorporated herein in their entireties byreference thereto.

2. SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 11, 2016, isnamed 381493-259US(144357)_SL.txt and is 104,856 bytes in size.

3. FIELD

The present application pertains to, among other things, anti-huLRRC15antibodies, antibody drug conjugates (“ADCs”), compositions includingthe ADCs, methods of making the ADCs, and methods of using the ADCs totreat cancers.

4. BACKGROUND

Cancer therapies comprise a wide range of therapeutic approaches,including surgery, radiation, and chemotherapy. While the oftencomplementary approaches allow a broad selection to be available to themedical practitioner to treat the cancer, existing therapeutics sufferfrom a number of disadvantages, such as a lack of selectivity oftargeting cancer cells over normal, healthy cells, and the developmentof resistance by the cancer to the treatment.

Recent approaches based on targeted therapeutics, which interfere withcellular processes of cancer cells preferentially over normal cells,have led to chemotherapeutic regimens with fewer side effects ascompared to non-targeted therapies such as radiation treatment.Nevertheless, cancers treated with targeted therapeutics may stilldevelop resistance. For example, resistance to bevacizumab, a monoclonalantibody therapeutic that targets VEGF-positive cancer cells, has beenreported in some colorectal cancers (Mesange et al. Oncotarget 2014;5(13): 4709-4721).

A mechanism for treatment resistance is believed to be the formation byactivated fibroblasts (e.g., cancer associated fibroblasts (CAFs),mesenchymal stem cells (MSCs)) in the tumor microenvironment whichprevents cancer drugs from physically reaching the cancer cells (KalluriR., Nature Reviews Cancer 2016; 16: 582-598). In addition, thefibroblast-mediated stromal barrier is understood to give rise to animmunosuppressive environment that can prevent immune effector cellsfrom penetrating deep into the tumor and targeting cancer cells (Turley,S. J. Nature Reviews Immunology 2015; 15:669-682). Hence, a cancer drugthat targets these stromal fibroblast populations within the tumormicroenvironment would complement existing therapeutic strategies andmay overcome chemoresistance and increase sensitivity to immune mediatedtherapies.

Another source of treatment resistance is thought to be the plasticityof cancer cells. For example, the plasticity of cancer cells betweenepithelial and mesenchymal states has been implicated as a mechanism forthe generation of cancer stem cells, which can initiate tumors, as wellas serve as a starting point for metastasis. See Ye, X.; Weinberg, R. A.Trends in Cell Biology 2015; 25 (11): 675-686. Further, mesenchymalcancer cells have been reported to be resistant to standard cancertherapies, such as docetaxel. See Singh and Settleman. Oncogene 2010;29(34): 4741-4751; Ippolito, L. et al. Oncotarget 2016; 7 (38):61890-61904. A cancer therapy that is effective against these resistantcancer cells would complement existing therapeutic approaches.

Thus, cancer therapeutics that spare normal cells and are less prone todeveloping clinical resistance would provide additional options fortreating cancer, such as by augmenting existing standard of careregimens.

5. SUMMARY

Human LRRC15 (leucine-rich repeat-containing protein 15) is a cellsurface protein that has been reported to exist in two isoforms: onecontaining 587 amino acids (SEQ ID NO:1; NP_001128529.2) and anothercontaining 581 amino acids (SEQ ID NO:3; NP_570843.2) that is truncatedat its N-terminus as compared to the longer isoform of SEQ ID NO:1. Theamino acid sequences of both isoforms are illustrated in FIGS. 1A-1D.For ease of discussion, human LRRC15 is abbreviated herein as“huLRRC15.” This abbreviation is intended to refer to either isoform. Ininstances where a specific isoform is intended, the abbreviations“huLRRC151” and “huLRRC15s” for the longer isoform of SEQ ID NO:1 andshorter isoform of SEQ ID NO:3, respectively, are used.

Referring to FIGS. 1C-1D (SEQ ID NO:3), huLRRC15 comprises anextracellular domain (“ECD”) spanning residues 22 to 538, atransmembrane domain (“TMD”) spanning residues 539 to 559, and anintracellular domain (“ICD”) spanning residues 560 to 581. The leadersequence of huLRRC15, illustrated in FIGS. 1A-1B (SEQ ID NO:1), is shownin bold text and the transmembrane domain underlined, thereby indicatingthe ECD, TMD and ICD of their isoforms. Referring again to FIGS. 1C-1D(SEQ ID NO:3), the ECD contains a proteolytic cleavage siteapproximately between residues Arg⁵²⁷ and Ser⁵²⁸, the cleavage of whichresults in shedding of the portion of the ECD spanning approximatelyresidues 24-527 (“shed ECD” or “sECD”) from the cell surface and intothe blood stream. huLRRC15 is highly expressed in the stromalmicroenvironment (and specifically on cancer-associated fibroblasts) ofseveral solid tumors (see, e.g., Example 4 and FIG. 7), and exhibitslimited expression in normal tissue types (see, e.g., Example 5 and FIG.8). It is also expressed on certain cancer cells per se (e.g., sarcomas,melanomas and glioblastomas, data not shown). Data presented hereindemonstrate, for the first time, that antibody drug conjugates (“ADCs”)that specifically target huLRRC15 exhibit potent antitumor effects, bothalone and in combination with other targeted and non-targeted antitumortherapies, against solid tumors in which huLRRC15 is expressed in thetumor stromal microenvironment, but not on the cancer cells per se((referred to herein as “huLRRC15 stromal(+)/cancer(−) tumors”)). Datademonstrating in vivo anti-tumor efficacy of anti-huLRRC15 ADCsadministered as monotherapy are provided in Example 10 and FIGS.13A-13C. While not intending to be bound by any theory of operation, itis believed that this potent antitumor effect is mediated, at least inpart, via a targeted bystander killing effect (see, e.g., Example 12 andFIGS. 15A-15I and 17B), although direct killing of stromal cellsexpressing huLRRC15 may also play a role. This potent antitumor activityis surprisingly observed with anti-LRRC15 ADCs that specifically bindthe portion of the huLRRC15 ECD domain that is shed from the cellsurface, and demonstrates for the first time that such anti-LRRC15 ADCsmay be used as therapeutically for the treatment of huLRRC15stromal(+)/cancer(−) tumors.

Accordingly, in one aspect, the present disclosure provides ADCs thatspecifically bind huLRRC15 (“anti-huLRRC15 ADCs”). The anti-huLRRC15ADCs comprise cytotoxic and/or cytostatic agents linked by way oflinkers to an antigen binding moiety that specifically binds huLRRC15 ata portion of the ECD that is shed from the cell surface. The antigenbinding moiety may be any moiety capable of specifically bindinghuLRRC15. In some embodiments, the antigen binding moiety is an antibodyand/or an antibody antigen binding fragment.

Antibodies and/or binding fragments composing the anti-huLRRC15 ADCsgenerally comprise a heavy chain comprising a variable region (V_(H))having three complementarity determining regions (“CDRs”) referred toherein (in N→C order) as V_(H) CDR#1, V_(H) CDR#2, and V_(H) CDR#3, anda light chain comprising a variable region (V_(L)) having threecomplementarity determining regions referred to herein (in N→C order) asV_(L) CDR#1, V_(L) CDR#2, and V_(L) CDR#3. The amino acid sequences ofexemplary CDRs, as well as the amino acid sequence of the V_(H) andV_(L) regions of the heavy and light chains of exemplary anti-huLRRC15antibodies and/or binding fragments that can be included in antigenbinding moieties composing the anti-huLRRC15 ADCs are provided herein.Specific embodiments of anti-huLRRC15 ADCs include, but are not limitedto, those that comprise antibodies and/or binding fragments that includethese exemplary CDRs and/or V_(H) and/or V_(L) sequences, as well asantibodies and/or binding fragments that compete for binding huLRRC15with such antibodies and/or binding fragments.

Antibodies may be in the form of full-length antibodies, bispecificantibodies, dual variable domain antibodies, multiple chain or singlechain antibodies, surrobodies (including surrogate light chainconstruct), single domain antibodies, camelized antibodies, scFv-Fcantibodies, and the like. They may be of, or derived from, any isotype,including, for example, IgA (e.g., IgA₁ or IgA₂), IgD, IgE, IgG (e.g.,IgG₁, IgG₂, IgG₃ or IgG₄), IgM, or IgY. In some embodiments, theanti-huLRRC15 antibody is an IgG (e.g., IgG₁, IgG₂, IgG₃ or IgG₄).Antibodies may be of human or non-human origin. Examples of non-humanorigin include, but are not limited to, mammalian origin (e.g., simians,rodents, goats, and rabbits) or avian origin (e.g., chickens). Inspecific embodiments, antibodies composing the anti-huLRRC15 ADCs aresuitable for administration to humans, such as, for example, humanizedantibodies and/or fully human antibodies.

Antibody antigen binding fragments composing the anti-huLRRC15 ADCs mayinclude any fragment of an antibody capable of specifically bindinghuLRRC15. Specific examples of antibody antigen binding fragments thatmay be included in the anti-huLRRC15 ADCs include, but are not limitedto, Fab, Fab′, (Fab′)₂, Fv and scFv.

Antibodies and/or binding fragments composing the anti-huLRRC15 ADCs mayinclude modifications and/or mutations that alter the properties of theantibodies and/or fragments, such as those that increase half-life,increase or decrease ADCC, etc., as is known in the art.

For therapeutic uses, it may be desirable to utilize anti-huLRRC15 ADCsthat bind huLRRC15 with an affinity of at least 100 nM. Accordingly, insome embodiments, the anti-huLRRC15 ADCs comprise an anti-huLRRC15antibody and/or anti-huLRRC15 binding fragment that binds huLRRC15 withan affinity of at least about 100 nM, or even higher, for example, atleast about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20nM, 15 nM, 10 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.1 nM, 0.01nM, or greater. Affinity of anti-huLRRC15 antibodies and/or bindingfragments can be determined using techniques well known in the art ordescribed herein, such as for example, ELISA, isothermal titrationcalorimetry (ITC), surface plasmon resonance, flow cytometry, orfluorescent polarization assay.

The cytotoxic and/or cytostatic agents composing the anti-huLRRC15 ADCsmay be any agents known to inhibit the growth and/or replication of,and/or kill cells. Numerous agents having cytotoxic and/or cytostaticproperties are known in the literature. Non-limiting examples of classesof cytotoxic and/or cytostatic agents include, by way of example and notlimitation, cell cycle modulators, apoptosis regulators, kinaseinhibitors, protein synthesis inhibitors, alkylating agents, DNAcross-linking agents, intercalating agents, mitochondria inhibitors,nuclear export inhibitors, topoisomerase I inhibitors, topoisomerase IIinhibitors, RNA/DNA antimetabolites and antimitotic agents.

As will be discussed in more detail in the Detailed Description, andwhile not intending to be bound by any particular theory of operation,data included herein demonstrate that anti-huLRRC15 ADCs exert potentanti-tumor activities mediated, at least in part, by a targetedbystander effect. For example, it is demonstrated herein that ananti-huLRRC15 ADC comprising the microtubule inhibitor monomethylauristatin E (“MMAE”) linked to an anti-huLRRC15 antibody by way oflinkers cleavable by lysosomal enzymes potently inhibits and/or killshuLRRC15 stromal(+)/cancer(−) tumors, yet does not significantly inhibitor kill more slowly dividing huLRRC15-expressing stromal fibroblasts(see, e.g., Example 12 and FIGS. 15B-15I and 16). An anti-huLRRC15 ADCcomprising the microtubule inhibitor monomethyl auristatin F (“MMAF”)linked to an anti-huLRRC15 antibody by way of non-cleavable linkers hadpotent in vitro activity against cells expressing huLRRC15, but hadlittle efficacy against huLRRC15 stromal(+)/cancer(−) tumors in vivo(see, e.g., Example 12 and FIGS. 17A-17B). Together, these data indicatethat anti-huLRRC15 ADCs may be used to potently inhibit huLRRC15stromal(+)/cancer(−) tumors via two different mechanisms of action, or acombination of both mechanisms: a first mechanism in which the cytotoxicand/or cytostatic agents composing the anti-huLRRC15 ADCs are cytotoxicand/or cytostatic to the huLRRC15-expressing stromal cells per se,thereby disrupting and/or destroying the stromal microenvironmentcrucial to support and/or growth of the huLRRC15 stromal(+)/cancer(−)tumor; and a second mechanism in which the cytotoxic and/or cytostaticagents composing the anti-huLRRC15 ADCs are not necessarily cytotoxicand/or cytostatic to the huLRRC15-expressing stromal cells, but arecytostatic and/or cytotoxic to the rapidly dividing huLRRC15 cancer(−)cells. Skilled artisans will appreciate that for this latter mechanismof action, the cytotoxic and/or cytostatic agents composing theanti-huLRRC15 ADCs, once cleaved from the anti-huLRRC15 ADCs, should becapable of traversing cell membranes. For the former mechanism ofaction, the cytotoxic and/or cytostatic agents, once cleaved from theanti-huLRRC15 ADC, need not be capable of traversing cell membranes.Cytotoxic and/or cytostatic agents having hydrophobicities sufficient totraverse cell membranes such that they are useful for inhibiting and/orkilling tumors via a targeted bystander effect may be identified usingroutine methods known to those of skill in the art. Cytotoxic and/orcytostatic agents having hydrophobicities such that they are capable oftraversing cell membranes and permeating into cells are referred toherein as “cell-permeating cytotoxic and/or cytostatic agents.”

Skilled artisans will also appreciate that the above two mechanisms ofaction are not mutually exclusive, and that in some embodiments it maybe desirable to utilize anti-huLRRC15 ADCs capable of exerting antitumoractivity against huLRRC15 stromal(+)/cancer(−) tumors via bothmechanisms of action. As a specific example, such an anti-huLRRC15 ADCmay include a cell-permeating cytotoxic and/or cytostatic agent that iscytotoxic and/or cytostatic to both huLRRC15-expressing stromal cellsand huLRRC15 cancer(−) tumor cells linked to an anti-huLRRC15 antibodyby way of a cleavable linker. As another specific embodiment, such ananti-huLRRC15 ADC may include two different cytotoxic and/or cytostaticagents: a first that is cytotoxic and/or cytostatic to thehuLRRC15-expressing stromal cells (and optionally, but not necessarily,also cytotoxic and/or cytostatic to the huLRRC15 cancer(−) tumor cells);and a second that is cytotoxic and/or cytostatic to thehuLRRC15-expressing stromal cells. The first agent could be, but neednot be, a cell-permeating cytotoxic and/or cytostatic agent, and couldbe, but need not be, linked to the antigen binding moiety of theanti-huLRRC15 ADC by way of a cleavable linker. The second agent is acell-permeating cytotoxic and/or cytostatic agent and is linked to theantigen binding moiety of the anti-huLRRC15 ADC by way of a cleavablelinker.

In a specific embodiment, a cytotoxic and/or cytostatic agent composingan anti-huLRRC15 ADC is a cell-permeating antimitotic agent, such as,for example, an auristatin. Specific examples of cell-permeatingauristatins include, but are not limited to, dolastatin-10 andmonomethyl auristatin E (“MMAE”). In another specific embodiment, acytotoxic and/or cytostatic agent composing an anti-huLRRC15 ADC is acell-permeating DNA cross-linking agent, such as a cell-permeating minorgroove-binding DNA cross-linking agent. Specific examples ofcell-permeating DNA minor groove-binding agents include, but are notlimited to, pyrrolobenzodiazepines (“PBD”) and PBD dimers.

The linkers linking the cytotoxic and/or cytostatic agents to theantigen binding moiety of an anti-huLRRC15 ADC may be long, short,flexible, rigid, hydrophilic or hydrophobic in nature, or may comprisesegments that have different characteristics, such as segments offlexibility, segments of rigidity, etc. The linker may be chemicallystable to extracellular environments, for example, chemically stable inthe blood stream, or may include linkages that are not stable andrelease the cytotoxic and/or cytostatic agents in the extracellularmilieu. In some embodiments, the linkers include linkages that aredesigned to release the cytotoxic and/or cytostatic agents uponinternalization of the anti-huLRRC15 ADC within the cell. In somespecific embodiments, the linkers includes linkages designed to cleaveand/or immolate or otherwise breakdown specifically or non-specificallyinside cells. A wide variety of linkers useful for linking drugs toantigen binding moieties such as antibodies in the context of ADCs areknown in the art. Any of these linkers, as well as other linkers, may beused to link the cytotoxic and/or cytostatic agents to the antigenbinding moiety of the anti-huLRRC15 ADCs described herein.

The number of cytotoxic and/or cytostatic agents linked to the antigenbinding moiety of an anti-huLRRC15 ADC can vary (called the“drug-to-antibody ratio,” or “DAR”), and will be limited only by thenumber of available attachments sites on the antigen binding moiety andthe number of agents linked to a single linker. Typically, a linker willlink a single cytotoxic and/or cytostatic agent to the antigen bindingmoiety of an anti-huLRRC15 ADC. In embodiments of anti-huLRRC15 ADCswhich include more than a single cytotoxic and/or cytostatic agent, eachagent may be the same or different. As long as the anti-huLRRC15 ADCdoes not exhibit unacceptable levels of aggregation under the conditionsof use and/or storage, anti-huLRRC15 ADCs with DARs of twenty, or evenhigher, are contemplated. In some embodiments, the anti-huLRRC15 ADCsdescribed herein may have a DAR in the range of about 1-10, 1-8, 1-6, or1-4. In certain specific embodiments, the anti-huLRRC15 ADCs may have aDAR of 2, 3 or 4.

In some embodiments, the anti-huLRRC15 ADCs are compounds according tostructural formula (I):[D-L-XY]_(n)-Ab  (I)or salts thereof, where each “D” represents, independently of theothers, a cytotoxic and/or cytostatic agent; each “L” represents,independently of the others, a linker; “Ab” represents an anti-huLRRC15antigen binding moiety, such as an anti-huLRRC15 antibody or bindingfragment; each “XY” represents a linkage formed between a functionalgroup R^(x) on the linker and a “complementary” functional group R^(y)on the antigen binding moiety; and n represents the DAR of theanti-huLRRC15 ADC. In a specific exemplary embodiment, the anti-huLRRC15ADCs are compounds according to structural formula (I) in which each “D”is the same and is either a cell-permeating auristatin (for example,dolastatin-10 or MMAE) or a cell-permeating minor groove-binding DNAcross-linking agent (for example, a PBD or a PBD dimer); each “L” is thesame and is a linker cleavable by a lysosomal enzyme; each “XY” is alinkage formed between a maleimide and a sulfydryl group; “Ab” is anantibody comprising six CDRs corresponding to the six CDRs of antibodyhuM25, huAD208.4.1, huAD208.12.1, huAD208.14.1, hu139.10, muAD210.40.9or muAD209.9.1, or an antibody that competes for binding huLRRC15 withsuch an antibody; and n is 2, 3 or 4. In a specific embodiment of thisexemplary embodiment or the anti-huLRRC15 ADCs of structural formula(I), “Ab” is a humanized antibody, for example, a humanized antibodycomprising V_(H) and V_(L) chains corresponding to the V_(H) and V_(L)chains of antibody huM25, huAD208.4.1, huAD208.12.1, huAD208.14.1, orhu139.10. In another specific embodiment of this exemplary embodiment orthe anti-huLRRC15 ADCs of structural formula (I), Ab is a humanizedantibody selected from huM25, huM25-S239C, huAD208.4.1,huAD208.4.1-S239C, huAD208.12.1, huAD208.14.1 and hu139.10.

In another aspect, the present disclosure provides compositionsincluding the anti-huLRRC15 ADCs. The compositions generally compriseone or more anti-huLRRC15 ADCs as described herein, and/or saltsthereof, and one or more excipients, carriers or diluents. Thecompositions may be formulated for pharmaceutical use, or other uses. Inone specific embodiment, the composition is formulated forpharmaceutical use and comprises an anti-huLRRC15 ADC according tostructural formula (I) or any of the specific exemplary embodimentsthereof, and one or more pharmaceutically acceptable excipients,carriers or diluents.

Compositions formulated for pharmaceutical use may be packaged in bulkform suitable for multiple administrations, or may be packaged in theform of unit doses suitable for a single administration. Whetherpackaged in bulk or in the form of unit doses, the composition may bepresented in dry form, such as a lyophilate, or in liquid form. Unitdosage liquid compositions may be conveniently packaged in the form ofsyringes pre-filled with a quantity of anti-huLRRC15 ADC suitable for asingle administration.

Also provided are unconjugated anti-huLRRC15 antibodies and/or bindingfragments. Such antibodies may be used in a variety of contexts in vitroand in vivo, including, by way of example and not limitation, ascellular stains for biological assays and as diagnostic agents tomonitor treatment of huLRRC15 stromal(+)/cancer(−) tumors, whether thetreatment is with anti-huLRRC15 ADCs or other agents, or a combinationof agents. The anti-huLRRC15 antibodies and/or binding fragments may belabeled with moieties to aid detection in diagnostic or other assays, ormay be unlabeled. Suitable labels include, by way of example and notlimitation, isotopic labels, fluorescent labels, chemiluminescentlabels, substrates for enzymes or other binding molecules, etc.Exemplary embodiments of anti-huLRRC15 antibodies and/or bindingfragments include the various exemplary anti-huLRRC15 antibodies and/orbinding fragments described herein in connection with the anti-huLRRC15antibodies and/or binding fragments described herein in connection withthe anti-huLRRC15 ADCs.

Also provided are polynucleotides encoding antigen binding moieties (forexample antibodies and/or binding fragments) that compose theanti-huLRRC15 ADCs described herein, host cells transformed ortransfected with the polynucleotides, and compositions and methodsuseful for making the various anti-huLRRC15 ADCs described herein.

As noted above, anti-huLRRC15 ADCs including cell-permeating cytotoxicand/or cytostatic agents exhibit potent antitumor activity againsthuLRRC15 stromal(+)/cancer(−) tumors that is believed to be mediated, atleast in part, by a targeted bystander killing effect, and that thispotent antitumor activity is observed with anti-huLRRC15 ADCs thatspecifically bind the portion of the huLRRC15 ECD that can be shed fromthe cell surface. This is surprising, as the shed ECD is available as a“sink” or “decoy” for the anti-huLRRC15 ADCs, thereby interfering withtheir ability to bind and become internalized into huLRRC15-expressingcells. Data provided herein demonstrate, for the first time, that ADCstargeting huLRRC15 may be used therapeutically for the treatment ofhuLRRC15 stromal(+)/cancer(−) tumors.

Accordingly, in another aspect, the present disclosure provides methodsof using anti-huLRRC15 ADCs therapeutically for the treatment ofhuLRRC15 stromal(+)/cancer(−) tumors. The methods generally involveadministering to a human patient having a huLRRC15 stromal(+)/cancer(−)tumor an amount of an anti-huLRRC15 ADC sufficient to providetherapeutic benefit. Human LRRC15 stromal(+)/cancer(−) tumors that canbe beneficially treated with anti-huLRRC15 ADCs include, but are notlimited to breast cancers, lung cancers (for example, non-small celllung cancers), head and neck cancers, pancreatic cancers, colorectalcancers, bladder cancers, liver cancers, and ovarian cancers. The cancermay be newly diagnosed and naïve to treatment, or may be relapsed,refractory, or relapsed and refractory, or a metastatic form of ahuLRRC15 stromal(+)/cancer(−) tumor.

The anti-huLRRC15 ADCs may be administered as single therapeutic agents(monotherapy) or adjunctive to, or with other anti-cancer treatmentsand/or therapeutic agents, typically but not necessarily those used totreat the type of huLRRC15 stromal(+)/cancer(−) tumors being treated.Indeed, data presented herein demonstrate that tumors that re-grow posttreatment when treated with currently available agents, or that exhibitresistance to other targeted or non-targeted chemotherapies, retainsensitivity to anti-huLRRC15 ADCs (see, e.g., Example 14 and FIGS.20A-20E). While not wishing to be bound by theory, one possiblemechanism of action of the anti-huLRRC15 ADCs of the disclosure may bethe killing of cancer cells that exhibit mesenchymal-like propertieswhich lend them resistance to standard therapies. Accordingly, theanti-huLRRC15 ADCs described herein provide significant benefits overcurrent targeted and non-targeted approaches toward the treatment ofhuLRRC15 stromal(+)/cancer(−) tumors. Adjunctive therapies and/ortherapeutic agents typically will be used at their approved dose, routeof administration, and frequency of administration, but may be used atlower dosages and/or less frequently. When administered as monotherapy,the anti-huLRRC15 ADC will typically be administered on a schedule thatbalances patient convenience and therapeutic benefit. It is contemplatedthat anti-huLRRC15 ADCs administered once a week, once every two weeks,once every three weeks, once every four weeks, once every five weeks,once every six weeks, once every seven weeks or once every eight weekswill provide therapeutic benefit, although more or less frequentadministration may be beneficial. When administered adjunctive to orwith another therapy and/or agent, the anti-huLRRC15 ADC may beadministered before, after or concurrently with the other therapy oragent.

The anti-huLRRC15 ADCs may be administered via a variety of routes ormodes of administration, including but not limited to, intravenousinfusion and/or injection and subcutaneous injection. The amountadministered will depend upon the route of administration, the dosingschedule, the type of cancer being treated, the stage of the cancerbeing treated, and other parameters such as the age and weight of thepatient, as is well known in the art. Specific exemplary dosingschedules expected to provide therapeutic benefit are provided in theDetailed Description. Generally, an amount of anti-huLRRC15 ADC in therange of about 0.01 to 10 mg/kg when administered intravenously on aweekly basis from once weekly to and including once every eight weeks isexpected to provide therapeutic benefit.

6. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D provide the amino acid sequences of the two reportedisoforms of human LRRC15, and the corresponding DNA sequences encodingeach. FIGS. 1A-1B show the encoding DNA sequence (SEQ ID NO:2) and aminoacid sequence (SEQ ID NO:1) for longer huLRRC15 isoform 1: FIG. 1Acorresponds to coding DNA residues 1-1020; FIG. 1B corresponds to codingDNA residues 1021-1761. FIGS. 1C-1D show the encoding DNA sequence (SEQID NO:4) and amino acid sequence (SEQ ID NO:3) for shorter huLRRC15isoform 2: FIG. 1C corresponds to coding DNA residues 1-1020; FIG. 1Dcorresponds to coding DNA residues 1021-1743. The predicted signalpeptide is indicated in bold italics; the predicted protease cleavagesite is boxed; and the predicted transmembrane domain is underlined.

FIG. 2A provides the amino acid sequences of the V_(H) chains ofantibodies huM25, huAD208.4.1, huAD208.12.1, huAD208.14.1, hu139.10,muAD210.40.9 and muAD209.9.1. The CDRs are underlined.

FIG. 2B provides the amino acid sequences of the V_(L) chains ofantibodies huM25, huAD208.4.1, huAD208.12.1, huAD208.14.1, hu139.10,muAD210.40.9 and muAD209.9.1. The CDRs are underlined.

FIG. 3A provides the amino acid sequence of the heavy chain of antibodyhuM25 (SEQ ID NO:18). The CDRs are underlined, and the constant region(Fc gamma) is italicized. Linear amino acid sequence numbering is shown.

FIG. 3B provides the amino acid sequence of the light chain of antibodyhuM25 (SEQ ID NO:19). The CDRs are underlined, and the constant region(kappa) is italicized. Linear amino acid sequence numbering is shown.

FIG. 4 provides a graph illustrating the ability of various exemplaryanti-huLRRC15 antibodies having CDRs that may be included inanti-huLRRC15 ADCs to bind to cells expressing huLRRC15.

FIG. 5 provides a graph illustrating the ability of various exemplaryanti-huLRRC15 antibodies having CDRs that may be included inanti-huLRRC15 to bind the extracellular domain (ECD) of huLRRC15.

FIGS. 6A and 6B together provide results of antibody competition assaysillustrating that exemplary anti-huLRRC15 antibodies having CDRs thatmay be included in anti-huLRRC15 ADCs bind different epitopes onhuLRRC15. FIG. 6A shows binding competition of muM25, huM25, hu139.10 orisotype antibodies against muM25-AF488; FIG. 6B shows bindingcompetition of muM25, muAD208.4.1, muAD208.12.1, muAD208.14.1,muAD209.9.1 or isotype antibodies against muM25-AF488.

FIG. 7 provides pictures of immunohistochemistry (IHC) andimmunofluorescence (IF) stained tissues illustrating that huLRRC15 ishighly expressed in the stromal microenvironment of various solidtumors.

FIG. 8 provides pictures of IHC stained tissues illustrating thathuLRRC15 is either not expressed, or only minimally expressed, onvarious normal tissue types.

FIG. 9A depicts huLRRC15 expression levels as measured by Western blotanalysis in a patient-derived breast cancer-associated fibroblast (CAF)lysate, or in commercially available patient-derived mesenchymal stemcell (MSC) lysates from bone marrow, in the absence (−) or presence (+)of 10 ng/mL TGFβ. Anti-huLRRC15 antibody used for detection wasmuAD210.40.9.

FIGS. 9B-9C depict huLRRC15 expression in commercial mesenchymal stemcell (MSC) lines, with and without 10 ng/mL TGFβ, as measured by flowcytometry: FIG. 9B depicts huLRRC15 expression as compared with isotypein human BM-MSC cells (Lonza); FIG. 9C depicts huLRRC15 expression ascompared with isotype in murine Balb/c BM-MSC cells (Cyagen).Anti-huLRRC15 antibody used for detection was AF647-labeled huM25.

FIG. 10 depicts expression levels of huLRRC15, Snail, TCF8/ZEB1,N-Cadherin, E-Cadherin, and GAPDH as determined by Western blot analysisof A549 lung cancer or PANC1 pancreatic cancer cells with 5 daytreatment of 10 ng/mL TGFβ, StemXVivo™ EMT Inducing Media Supplement(R&D Systems, “EMT Kit”), or control. Anti-LRRC15 antibody used fordetection was muAD210.40.9; all other antibodies were obtained from theEMT Antibody Panel (Cell Signaling Technology).

FIG. 11A depicts protein expression as determined by fluorescence inA549 lung cancer or PANC1 pancreatic cancer cells after 5 day treatmentof 10 ng/mL TGFβ, StemXVivo™ EMT Inducing Media Supplement (R&D Systems,“EMT Kit”), or control. Fluorescence using AF647-labeled isotype(“Isotype-AF647”), AF647-labeled huM25 (“huM25-AF647”), PE-labeledisotype (“Isotype-PE”), and PE-labeled anti-EpCAM (“anti-EPCAM-PE”)antibodies is shown.

FIG. 11B depicts microscopy images of A549 cells after continuedtreatment of 10 ng/mL TGFβ over 9 days (upper left, “continuedtreatment”), or treatment with 10 ng/mL TGFβ for 5 days followed bywashout and culturing in the absence of TGFβ for an additional 4 days(lower left, “discontinued treatment”). Flow cytometry (middle and rightgraphs) depicts huLRRC15 (upper and lower middle) and EpCAM (upper andlower right) levels after continued treatment, discontinued treatment,or control. Anti-huLRRC15 antibody huM25-AF647 was used in the analysis.

FIG. 11C depicts protein expression as determined by fluorescence inA549 lung cancer (left graphs) or PANC1 pancreatic cancer cells (rightgraphs) after 9 day continued treatment of 10 ng/mL TGFβ or StemXVivo™EMT Inducing Media Supplement (R&D Systems, “EMT Kit”), or treatmentwith TGFβ or EMT Kit for 5 days followed by washout and culturing for anadditional 4 days. Top panels depict huLRRC15 levels; bottom panelsdepict EpCAM levels.

FIG. 12 provides chromatograms of ADC preparations. The top panelillustrates a chromatographic resolution of a conjugation carried outaccording to Example 8. Several peaks are present, corresponding toantibodies having zero (“DAR0”), two (“DAR2”), four (“DAR4”), six(“DAR6”) and eight (“DAR8”) linked cytostatic and/or cytotoxic agents.The preparation has an average DAR of 4. This crude average DAR4preparation was subjected to hydrophobic interaction chromatography toisolate the peak corresponding to DAR2. The chromatogram of theresultant preparation enriched in DAR2 (referred to herein as an “E2”preparation) is shown in the bottom panel. The enriched E2 ADCpreparation is approximately 98% pure in ADCs having a DAR of 2.

FIGS. 13A-13C provide graphs demonstrating the potent in vivo efficacyof exemplary anti-huLRRC15 ADCs against a variety ofstromal(+)/cancer(−) tumors. In the graphs, the arrows indicate dosingdays. Also shown are pictures illustrating LRRC15 expression on stromalcells as assessed by IHC in an untreated xenograft tumor of 200-800 mm³in volume, representative for each xenograft model. FIG. 13Ademonstrates in vivo activity of huM25-vcMMAE-E2 in EBC-1 xenografts;FIG. 13B demonstrates in vivo activity of huM25-vcMMAE-E2 in HPAF-IIxenografts; and FIG. 13C demonstrates in vivo activity ofhuM25-vcMMAE-DAR4, huAD208.4.1-vcMMAE-DAR4 and huAD208.14.1-vcMMAE-DAR4in EBC-1 xenografts.

FIG. 13D provides a graph illustrating that huM25-vcMMAE-DAR4 is notactive in vivo against xenografts generated with HCC-827-ER despite highhuLRRC15 expression in stroma. Arrows indicate dosing days. Also shownis a representative IHC picture illustrating LRRC15 expression onstromal cells for this xenograft model.

FIG. 14 provides a graph demonstrating that tumors that regrow followingtreatment with exemplary anti-huLRRC15 ADC huM25-vcMMAE-DAR4 retainexpression of huLRRC15, and therefore retain sensitivity toanti-huLRRC15 ADCs. Arrows represent dosing days. Also shown arepictures illustrating LRRC15 expression on stromal cells, as assessed byIHC, in an untreated xenograft tumor immediately prior to treatment (Day0) or following treatment and regrowth (Day 70).

FIGS. 15A-15I provide graphs and pictograms demonstrating that theanti-tumor activity of exemplary anti-huLRRC15 ADC huM25-vcMMAE-E2against huLRRC15 stromal(+)/cancer(−) tumors is mediated at least inpart by a bystander killing effect. Also shown is a representative IHCpicture illustrating LRRC15 expression on stromal cells for thisxenograft model. FIG. 15A depicts the timing for removal of tumors forex vivo analysis. FIGS. 15B-15E depict ex vivo flow cytometry data ofEBC-1 tumors treated with huM25-vcMMAE-E2 or huM25-mcMMAF-E2: FIG. 15Bshows EPCAM expression; FIG. 15C shows FAP expression; FIG. 15D showsCD11c expression; and FIG. 15E shows F4/80 expression. FIG. 15F depictsmicroscopic analysis of ex vivo EBC-1 tumors treated withhuM25-vcMMAE-E2. FIG. 15G depicts immunohistochemical (IHC) staining ofEBC-1 tumors showing LRRC15 expression in untreated tumors (left ofdashed line), or phospho-histone-H3 (pHH3) staining of untreated tumorsor tumors treated with 6 mg/kg of either isotype-vcMMAE-E2 orhuM25-vcMMAE-E2 (right of dashed line). FIG. 15H shows quantitatedimmunohistochemical analysis of percentage phospho-histone-H3 (% pHH3)positive cells vs. days post sizematch in untreated EBC-1 tumors ortumors treated with 6 mg/kg huM25-vcMMAE-E2. FIG. 15I shows expressionof CD45 (left) or F4/80 (right) by staining of EBC-1 tumors after 11days post-treatment with 6 mg/kg of isotype-vcMMAE-E2 (top panels) orhuM25-vcMMAE-E2 (bottom panels).

FIG. 16 provides data demonstrating that stromal fibroblasts are lessproliferative than cancer cells across a panel of different tumor types,using Ki67 expression as a marker of proliferation.

FIGS. 17A and 17B compare the anti-tumor activities of huM25-vcMMAE-E2and huM25-mcMMAF-E2 in vitro (FIG. 17A) and in vivo (FIG. 17B). In FIG.17B, arrows represent dosing days. Also shown is a representative IHCpicture illustrating LRRC15 expression on stromal cells for thisxenograft model.

FIGS. 18A and 18B provide data demonstrating that, on an equivalent drugbasis, E2 enriched preparations of anti-huLRRC15 ADCs are bettertolerated than higher loaded E4 preparations when assessed by survival(FIG. 15A) or weight loss (FIG. 15B). Arrows represent dosing days.

FIG. 19 provides data demonstrating that anti-huLRRC15 ADCs with E2 drugloading have efficacy comparable to anti-huLRRC15 ADCs having higherdrug loading, as assessed on an equivalent drug basis. Arrows representdosing days. Also shown is a representative IHC picture illustratingLRRC15 expression on stromal cells for this xenograft model.

FIGS. 20A-20E provide data demonstrating that anti-huLRRC15 ADCs aresuperior to current standard of care agents such as carboplatin orerlotinib (FIG. 20A); erlotinib or cetuximab (FIG. 20B); carboplatin orerlotinib (FIG. 20C); doxorubicin (FIG. 20D); and gemcitabine (FIG. 20E)in in vivo models of efficacy. Arrows represent dosing days. Also shownare representative IHC pictures illustrating LRRC15 expression onstromal cells for each of these xenograft models.

FIGS. 21A-21E provide data demonstrating that anti-huLRRC15 ADCs areeffective when administered adjunctive to current standard of carecytotoxic anti-cancer therapies. FIG. 21A depicts the anti-tumoractivity of huM25-vcMMAE-E2 with gemcitabine (Gem) in HPAF-II; FIG. 21Bdepicts the anti-tumor activity of huM25-vcMMAE-E2 with gemcitabine(Gem) in EBC-1; FIG. 21C depicts the anti-tumor activity ofhuM25-vcMMAE-DAR4 with docetaxel; FIG. 21D depicts the anti-tumoractivity of huM25-vcMMAE-E2 with radiation; and FIG. 21E depicts theanti-tumor activity of huM25-vcMMAE-E2 with carboplatin. Arrowsrepresent dosing days. Also shown are representative IHC picturesillustrating LRRC15 expression on stromal cells for each of thesexenograft models.

FIGS. 22A-22C provide data demonstrating that anti-huLRRC15 ADCs areeffective when administered adjunctive to targeted anti-cancertherapies. FIG. 22A depicts the anti-tumor activity of huM25-vcMMAE-E2with erlotinib; FIG. 22B depicts the anti-tumor activity ofhuM25-vcMMAE-E2 with cetuximab; and FIG. 22C depicts the anti-tumoractivity of huM25-vcMMAE-E2 with an anti-PD-1 antibody. Arrows representdosing days. Also shown are representative IHC pictures illustratingLRRC15 expression on stromal cells for each of these xenograft models.

FIGS. 23A-23D show in vitro data for exemplary anti-huLRRC15 ADCscomprising a minor groove binding pyrrolobenzodiazepine (PBD) cytotoxicand/or cytostatic agent. FIG. 23A shows in vitro cell killing in LRRC15transfected 3T12 cells by isotype-S239C-PBD-E2 (solid line) orhuM25-S239C-PBD-E2 (dashed line); FIG. 23B shows in vitro cell killingin human BM-MSC (Lonza) mesenchymal stem cells in the presence of 10ng/mL TGFβ by isotype-PBD-DAR2 or huAD208.4.1-PBD-DAR2; FIG. 23C showsin vitro cell killing in murine Balb/c BM-MSC (Cyagen) mesenchymal stemcells in the presence of 10 ng/mL TGFβ by isotype-PBD-DAR2 orhuAD208.4.1-PBD-DAR2; FIG. 23D shows in vitro cell killing in A549 lungcancer cells (top) or A549 cells that have undergone epithelial tomesenchymal transition (EMT) in the presence of 10 ng/mL TGFβ (bottom)by isotype-S239C-PBD-E2, huM25-S239C-PBD-E2, or huM25-S239C antibody. Inthe above graphs, the y-axis shows cell viability (%), and the x-axisshows antibody or ADC concentration in nM.

FIGS. 24A-24G provide data demonstrating that anti-huLRRC15 ADCscomprising a minor groove binding pyrrolobenzodiazepine (PBD) cytotoxicand/or cytostatic agent have potential anti-tumor activity againsthuLRRC15 stromal(+)/cancer(−) tumors in in vivo models: FIG. 24A depictsthe effect of a single dose of isotype-S239C-PBD-E2, huM25-S239C-PBD-E2or isotype antibody at 0.6 mg/kg on in vivo tumor volume (mm³) at 0-14days post sizematch of EBC-1 tumors. FIG. 24B depicts typical microscopyof tumor samples from experiment of FIG. 24A dosed with isotype antibody(left), isotype-S239C-PBD-E2 (middle), or huM25-S239C-PBD-E2 (right)stained for α-SMA. FIG. 24C depicts quantification of % α-SMA tumorpositivity across samples from experiment of FIG. 24A. FIG. 24D depictsF4/80 (left) or CD11c (right) expression in tumor samples fromexperiment of FIG. 24A, as measured by flow cytometry. FIG. 24E depictsthe effect of dosing huM25-S239C-PBD-E2 at 0.6, 0.3, or 0.1 mg/kg, or0.6 mg/kg isotype antibody, on in vivo tumor volume (mm³) at 0-80 dayspost sizematch of NCI-H1650 tumors. FIG. 24F depicts the effect ofdosing 0.3 mg/kg huM25-S239C-PBD-E2, or 0.3 mg/kg isotype-S239C-PBD-E2,on in vivo tumor volume (mm³) at 0-80 days post sizematch of NCI-H1650tumors. FIG. 24G shows effects of 0.6 mg/kg isotype-PBD-DAR2 orhuAD208.4.1-PBD-DAR2, or 12 mg/kg of huIgG1 isotype antibody, inNCI-H1650 tumors; FIG. 24H shows effects of 0.6 mg/kgisotype-vcMMAE-DAR2 or huAD208.4.1-PBD-DAR2, or 6 mg/kg of huIgG1isotype antibody, in EBC-1 tumors. Arrows represent dosing days. Alsoshown are representative IHC pictures illustrating LRRC15 expression onstromal cells for each of these xenograft models.

7. DETAILED DESCRIPTION

The present disclosure concerns antibody drug conjugates thatspecifically bind human LRRC15, compositions comprising the ADCs,anti-huLRRC15 antibodies and/or binding fragments that can comprise theADCs, polynucleotides encoding anti-huLRRC15 antibodies and/or bindingfragments that comprise the ADCs, host cells capable of producing theantibodies and/or binding fragments, methods and compositions useful formaking the antibodies, binding fragments and ADCs, and various methodsof using the ADCs.

As will be appreciated by skilled artisans, antibodies and/or bindingfragments are “modular” in nature. Throughout the disclosure, variousspecific embodiments of the various “modules” composing the antibodiesand/or binding fragments are described. As specific non-limitingexamples, various specific embodiments of V_(H) CDRs, V_(H) chains,V_(L) CDRs and V_(L) chains are described. It is intended that all ofthe specific embodiments may be combined with each other as though eachspecific combination were explicitly described individually.

The ADCs disclosed herein are also “modular” in nature. Throughout thedisclosure, various specific embodiments of the various “modules”composing the ADCs are described. As specific non-limiting examples,specific embodiments of antibodies, linkers, and cytotoxic and/orcytostatic agents that may compose the ADCs are described. It isintended that all of the specific embodiments described may be combinedwith each other as though each specific combination were explicitlydescribed individually.

It will also be appreciated by skilled artisans that the various ADCsdescribed herein may be in the form of salts, and in some specificembodiments, pharmaceutically acceptable salts. The ADCs of thedisclosure that possess a sufficiently acidic, a sufficiently basic, orboth functional groups, can react with any of a number of inorganicbases, and inorganic and organic acids, to form a salt. Alternatively,compounds that are inherently charged, such as those with a quaternarynitrogen, can form a salt with an appropriate counter ion, e.g., ahalide such as a bromide, chloride, or fluoride.

Acids commonly employed to form acid addition salts are inorganic acidssuch as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuricacid, phosphoric acid, and the like, and organic acids such asp-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid,etc. Base addition salts include those derived from inorganic bases,such as ammonium and alkali or alkaline earth metal hydroxides,carbonates, bicarbonates, and the like.

7.1. Abbreviations

The antibodies, binding fragments, ADCs and polynucleotides describedherein are, in many embodiments, described by way of their respectivepolypeptide or polynucleotide sequences. Unless indicated otherwise,polypeptide sequences are provided in N→C orientation; polynucleotidesequences in 5′→3′ orientation. For polypeptide sequences, theconventional three or one-letter abbreviations for the geneticallyencoded amino acids may be used, as noted in TABLE 1, below.

TABLE 1 Encoded Amino Acid Abbreviations Amino Acid Three LetterAbbreviation One-Letter Abbreviation Alanine Ala A Arginine Arg RAsparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu EGlutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV

Certain sequences are defined by structural formulae specifying aminoacid residues belonging to certain classes (e.g., aliphatic,hydrophobic, etc.). The various classes to which the genetically encodedamino acids belong as used herein are noted in TABLE 2, below. Someamino acids may belong to more than one class. Cysteine, which containsa sulfhydryl group, and proline, which is conformationally constrained,are not assigned classes.

TABLE 2 Encoded Amino Acid Classes Class Amino Acids Aliphatic A, I, L,V Aromatic F, Y, W Non-Polar M, A, I, L, V Polar N, Q, S, T Basic H, K,R Acidic D, E Small A, G

7.2. Definitions

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art.

7.3. Anti-huLRRC15 Antibody Drug Conjugates

In one aspect, the disclosure concerns antibody drug conjugates (“ADCs”)that specifically bind human LRRC15 isoform 1 (SEQ ID NO:1) or isoform 2(SEQ ID NO:3). The anti-huLRRC15 ADCs generally comprise ananti-huLRRC15 antigen binding moiety, for example an anti-huLRRC15antibody or binding fragment, having one or more cytotoxic and/orcytostatic agents linked thereto by way of one or more linkers.

7.3.1. Anti-huLRRC15 Antibodies and Binding Fragments

In specific exemplary embodiments, the antigen binding moiety is anantibody or an antibody antigen binding fragment. Antibodies and/orbinding fragments composing the anti-huLRRC15 ADCs specifically bindhuLRRC15 at a region of the extracellular domain (residues 22 to 527 ofSEQ ID NO:3) that is shed from the cell surface and into the bloodstream (“shed ECD” or “sECD”) following cleavage at a proteolyticcleavage site between Arg⁵²⁷ and Ser⁵²⁸ of SEQ ID NO:3.

As used herein, the term “antibody” (Ab) refers to an immunoglobulinmolecule that specifically binds to, or is immunologically reactivewith, a particular antigen-here, the sECD of huLRRC15. Antibodiescomprise complementarity determining regions (CDRs), also known ashypervariable regions, in both the light chain and heavy chain variabledomains. The more highly conserved portions of the variable domains arecalled the framework (FR). As is known in the art, the amino acidposition/boundary delineating a hypervariable region of an antibody canvary, depending on the context and the various definitions known in theart. Some positions within a variable domain may be viewed as hybridhypervariable positions in that these positions can be deemed to bewithin a hypervariable region under one set of criteria, while beingdeemed to be outside a hypervariable region under a different set ofcriteria. One or more of these positions can also be found in extendedhypervariable regions. The variable domains of native heavy and lightchains each comprise four FR regions, largely by adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen binding site of antibodies. See Kabat et al., Sequences ofProteins of Immunological Interest (National Institute of Health,Bethesda, Md. 1987). As used herein, numbering of immunoglobulin aminoacid residues is done according to the immunoglobulin amino acid residuenumbering system of Kabat et al. unless otherwise indicated.

Antibodies composing anti-huLRRC15 ADCs may be polyclonal, monoclonal,genetically engineered, and/or otherwise modified in nature, includingbut not limited to, chimeric antibodies, humanized antibodies, humanantibodies, primatized antibodies, single chain antibodies, bispecificantibodies, dual-variable domain antibodies, etc. In variousembodiments, the antibodies comprise all or a portion of a constantregion of an antibody. In some embodiments, the constant region is anisotype selected from: IgA (e.g., IgA₁ or IgA₂), IgD, IgE, IgG (e.g.,IgG₁, IgG₂, IgG₃ or IgG₄), IgM, and IgY. In specific embodiments,antibodies composing an anti-huLRRC15 ADC comprise an IgG₁ constantregion isotype.

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. A monoclonal antibodyis derived from a single clone, including any eukaryotic, prokaryotic,or phage clone, by any means available or known in the art. Monoclonalantibodies useful with the present disclosure can be prepared using awide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technologies, or a combinationthereof. In many uses of the present disclosure, including in vivo useof ADCs including anti-huLRRC15 antibodies in humans, chimeric,primatized, humanized, or human antibodies can suitably be used.

The term “chimeric” antibody as used herein refers to an antibody havingvariable sequences derived from a non-human immunoglobulin, such as arat or a mouse antibody, and human immunoglobulin constant regions,typically chosen from a human immunoglobulin template. Methods forproducing chimeric antibodies are known in the art. See, e.g., Morrison,1985, Science 229(4719):1202-7; Oi et al., 1986, BioTechniques4:214-221; Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporatedherein by reference in their entireties.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins that contain minimal sequences derived from non-humanimmunoglobulin. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin sequence. The humanizedantibody can also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin consensussequence. Methods of antibody humanization are known in the art. See,e.g., Riechmann et al., 1988, Nature 332:323-7; U.S. Pat. Nos.5,530,101; 5,585,089; 5,693,761; 5,693,762; and U.S. Pat. No. 6,180,370to Queen et al.; EP239400; PCT publication WO 91/09967; U.S. Pat. No.5,225,539; EP592106; EP519596; Padlan, 1991, Mol. Immunol., 28:489-498;Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994,Proc. Natl. Acad. Sci. 91:969-973; and U.S. Pat. No. 5,565,332, all ofwhich are hereby incorporated by reference in their entireties.

“Human antibodies” are antibodies having the amino acid sequence of ahuman immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries or from animals transgenic for one or morehuman immunoglobulin and that do not express endogenous immunoglobulins.Human antibodies can be made by a variety of methods known in the artincluding phage display methods using antibody libraries derived fromhuman immunoglobulin sequences. See U.S. Pat. Nos. 4,444,887 and4,716,111; and PCT publications WO 98/46645; WO 98/50433; WO 98/24893;WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741, each of which isincorporated herein by reference in its entirety. Human antibodies canalso be produced using transgenic mice which are incapable of expressingfunctional endogenous immunoglobulins but which can express humanimmunoglobulin genes. See, e.g., PCT publications WO 98/24893; WO92/01047; WO 96/34096; WO 96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793;5,916,771; and 5,939,598, which are incorporated by reference herein intheir entireties. In addition, companies such as Medarex (Princeton,N.J.), Astellas Pharma (Deerfield, Ill.), Amgen (Thousand Oaks, Calif.)and Regeneron (Tarrytown, N.Y.) can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above. Fully human antibodies that recognize aselected epitope can be generated using a technique referred to as“guided selection.” In this approach, a selected non-human monoclonalantibody, e.g., a mouse antibody, is used to guide the selection of acompletely human antibody recognizing the same epitope (see, Jespers etal., 1988, Biotechnology 12:899-903).

“Primatized antibodies” comprise monkey variable regions and humanconstant regions. Methods for producing primatized antibodies are knownin the art. See, e.g., U.S. Pat. Nos. 5,658,570; 5,681,722; and5,693,780, which are incorporated herein by reference in theirentireties.

Anti-huLRRC15 ADCs may comprise full-length (intact) antibody molecules,as well as antigen binding fragments that are capable of specificallybinding huLRRC15. Examples of antibody binding fragments include by wayof example and not limitation, Fab, Fab′, F(ab′)2, Fv fragments, singlechain Fv fragments and single domain fragments.

A Fab fragment contains the constant domain of the light chain and thefirst constant domain (CH₂) of the heavy chain. Fab′ fragments differfrom Fab fragments by the addition of a few residues at the carboxylterminus of the heavy chain CH₂ domain including one or more cysteinesfrom the antibody hinge region. F(ab′) fragments are produced bycleavage of the disulfide bond at the hinge cysteines of the F(ab′)₂pepsin digestion product. Additional chemical couplings of antibodyfragments are known to those of ordinary skill in the art. Fab andF(ab′)₂ fragments lack the Fc fragment of intact antibody, clear morerapidly from the circulation of animals, and may have less non-specifictissue binding than an intact antibody (see, e.g., Wahl et al., 1983, J.Nucl. Med. 24:316).

An “Fv” fragment is the minimum fragment of an antibody that contains acomplete target recognition and binding site. This region consists of adimer of one heavy and one light chain variable domain in a tight,non-covalent association (V_(H)-V_(L) dimer). It is in thisconfiguration that the three CDRs of each variable domain interact todefine an antigen binding site on the surface of the V_(H)-V_(L) dimer.Often, the six CDRs confer antigen binding specificity upon theantibody. However, in some instances even a single variable domain (orhalf of an Fv comprising only three CDRs specific for a target) may havethe ability to recognize and bind antigen, although at a lower affinitythan the entire binding site.

“Single-chain Fv” or “scFv” antibody binding fragments comprise theV_(H) and V_(L) domains of an antibody, where these domains are presentin a single polypeptide chain. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the scFv to form the desired structure for antigen binding.

“Single domain antibodies” are composed of a single V_(H) or V_(L)domains which exhibit sufficient affinity to huLRRC15. In a specificembodiment, the single domain antibody is a camelized antibody (See,e.g., Riechmann, 1999, Journal of Immunological Methods 231:25-38).

Antibodies composing the anti-huLRRC15 ADCs may also be bispecificantibodies. Bispecific antibodies are monoclonal, often human orhumanized, antibodies that have binding specificities for two differentepitopes on the same or different antigens. In the present disclosure,one of the binding specificities can be directed towards huLRRC15, theother can be for any other antigen, e.g., for a cell-surface protein,receptor, receptor subunit, tissue-specific antigen, virally derivedprotein, virally encoded envelope protein, bacterially derived protein,or bacterial surface protein, etc.

Antibodies composing anti-huLRRC15 ADCs may be derivatized. Derivatizedantibodies are typically modified by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein. Any of numerous chemical modifications may becarried out by known techniques, including, but not limited to, specificchemical cleavage, acetylation, formylation, metabolic synthesis oftunicamycin, etc. Additionally, the derivative may contain one or morenon-natural amino acids, e.g., using ambrx technology. See, e.g.,Wolfson, 2006, Chem. Biol. 13(10):1011-2.

Antibodies or binding fragments composing anti-huLRRC15 ADCs may beantibodies or fragments whose sequences have been modified to alter atleast one constant region-mediated biological effector function. Forexample, in some embodiments, an anti-huLRRC15 antibody may be modifiedto reduce at least one constant region-mediated biological effectorfunction relative to the unmodified antibody, e.g., reduced binding tothe Fc receptor (FcγR). FcγR binding may be reduced by mutating theimmunoglobulin constant region segment of the antibody at particularregions necessary for FcγR interactions (See, e.g., Canfield andMorrison, 1991, J. Exp. Med. 173:1483-1491; and Lund et al., 1991, J.Immunol. 147:2657-2662). Reducing FcγR binding may also reduce othereffector functions which rely on FcγR interactions, such asopsonization, phagocytosis and antigen-dependent cellular cytotoxicity(“ADCC”).

Antibodies or binding fragments composing anti-huLRRC15 ADCs may includemodifications that increase or decrease their binding affinities to theneonatal Fc receptor, FcRn, for example, by mutating the immunoglobulinconstant region segment at particular regions involved in FcRninteractions (see, e.g., WO 2005/123780). In particular embodiments, ananti-huLRRC15 antibody of the IgG class is mutated such that at leastone of amino acid residues 250, 314, and 428 of the heavy chain constantregion is substituted alone, or in any combinations thereof, such as atpositions 250 and 428, or at positions 250 and 314, or at positions 314and 428, or at positions 250, 314, and 428, with substitution atpositions 250 and 428 being a specific combination. For position 250,the substituting amino acid residue may be any amino acid residue otherthan threonine, including, but not limited to, alanine, cysteine,aspartic acid, glutamic acid, phenylalanine, glycine, histidine,isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine,arginine, serine, valine, tryptophan, or tyrosine. For position 314, thesubstituting amino acid residue may be any amino acid residue other thanleucine, including, but not limited to, alanine, cysteine, asparticacid, glutamic acid, phenylalanine, glycine, histidine, isoleucine,lysine, methionine, asparagine, proline, glutamine, arginine, serine,threonine, valine, tryptophan, or tyrosine. For position 428, thesubstituting amino acid residues may be any amino acid residue otherthan methionine, including, but not limited to, alanine, cysteine,aspartic acid, glutamic acid, phenylalanine, glycine, histidine,isoleucine, lysine, leucine, asparagine, proline, glutamine, arginine,serine, threonine, valine, tryptophan, or tyrosine. Specificcombinations of suitable amino acid substitutions are identified inTable 1 of U.S. Pat. No. 7,217,797, which is incorporated herein byreference. In yet further embodiments, the variant Fc domains have atleast one or more modifications that enhance the affinity to FcRn, e.g.,a modification of one or more amino acid residues 251-256, 285-290,308-314, 385-389, and 428-436 (e.g., M428L), or a modification atpositions 250 and 428 (e.g., T250Q/M428L). See, e.g., Hinton et al.,2004, J. Biol. Chem. 279 (8): 6213-6216; PCT Publication Nos. WO97/34631 and WO 02/060919. Such mutations increase binding to FcRn,which protects the antibody from degradation and increases itshalf-life.

An anti-huLRRC15 antibody and/or binding fragment may have one or moreamino acids inserted into one or more of its hypervariable regions, forexample as described in Jung & Plückthun, 1997, Protein Engineering10:9, 959-966; Yazaki et al., 2004, Protein Eng. Des Sel. 17(5):481-9;and U.S. Pat. App. No. 2007/0280931.

Post-translational modifications to the sequences of an antibodyincluded in an anti-huLRRC15 ADC may occur, such as cleavage of one ormore (e.g., 1, 2, 3, or more) amino acid residues on the C-terminal endof the antibody heavy chain.

Post-translational modifications of an antibody included in ananti-huLRRC15 ADC may include glycosylation. Common biantennarycomplexes can comprise a core structure having two N-acetylglucosamine(GlcNAc), three mannose, and two GlcNAc residues that are β-1,2 linkedto α-6 mannose and α-3 mannose to form two antennae. One or more fucose(Fuc), galactose (Gal), high mannose glycans Man-5 or Man-9, bisectingGlcNAc, and sialic acid including N-acetylneuraminic acid (NANA) orN-glycolylneuraminic acid (NGNA) residues may be attached to the core.N-linked glycoforms may include G0 (protein having a core biantennaryglycosylation structure), G0F (fucosylated G0), G0F GlcNAc, G1 (proteinhaving a core glycosylation structure with one galactose residue), G1F(fucosylated G1), G2 (protein having a core glycosylation structure withtwo galactose residues), and/or G2F (fucosylated G2).

Antibodies included in anti-huLRRC15 ADCs may have low levels of, orlack, fucose. Antibodies lacking fucose have been correlated withenhanced ADCC activity, especially at low doses of antibody. See Shieldset al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J.Biol. Chem. 278:3466-73. Methods of preparing fucose-less antibodiesinclude growth in rat myeloma YB2/0 cells (ATCC CRL 1662). YB2/0 cellsexpress low levels of FUT8 mRNA, which encodes α-1,6-fucosyltransferase,an enzyme necessary for fucosylation of polypeptides.

Anti-huLRRC15 antibodies and/or binding fragments with high affinity forhuLRRC15 may be desirable for therapeutic uses. Accordingly, the presentdisclosure contemplates ADCs comprising anti-huLRRC15 antibodies and/orbinding fragments having a high binding affinity to huLRRC15. Inspecific embodiments, the antibodies and/or binding fragments bindhuLRRC15 with an affinity of at least about 100 nM, but may exhibithigher affinity, for example, at least about 90 nM, 80 nM, 70 nM, 60 nM,50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 7 nM, 6 nM, 5 nM, 4 nM,3 nM, 2 nM, 1 nM, 0.1 nM, 0.01 nM, or even higher. In some embodiments,the antibodies bind huLRRC15 with an affinity in the range of about 1 pMto about 100 nM, or an affinity ranging between any of the foregoingvalues, such as but not limited to from about 0.01 to 100, 0.01 to 10,0.01 to 2, 0.1 to 100, 0.1 to 10, or 0.1 to 2 nM.

Affinity of antibodies and/or binding fragments for huLRRC15 can bedetermined using techniques well known in the art or described herein,such as for example, but not by way of limitation, ELISA, isothermaltitration calorimetry (ITC), surface plasmon resonance, flow cytometryor fluorescent polarization assays.

In some embodiments, an antibody and/or binding fragment composing ananti-huLRRC15 ADC comprises a V_(H) chain having three CDRs in whichV_(H) CDR#1, V_(H) CDR#2 and V_(H) CDR#3 have sequences selected fromtheir respective V_(H) CDR sequences in the table below:

CDR Sequence (N→C) Identifier huM25 V_(H) CDR#1 SYWIE SEQ ID NO: 10huAD208.4.1 V_(H) DYYIH SEQ ID NO: 20 CDR#1 huAD208.12.1 V_(H) NYWMH SEQID NO: 30 CDR#1 huAD208.14.1 V_(H) DYYIH SEQ ID NO: 40 CDR#1 hu139.10V_(H) CDR#1 SYGVH SEQ ID NO: 50 muAD210.40.9 V_(H) NYWLG SEQ ID NO: 60CDR#1 muAD209.9.1 V_(H) NFGMN SEQ ID NO: 70 CDR#1 huM25 V_(H) CDR#2EILPGSDTTNYNEKFKD SEQ ID NO: 11 huAD208.4.1 V_(H) LVYPYIGGTNYNQKFKG SEQID NO: 21 CDR#2 huAD208.12.1 V_(H) MIHPNSGSTKHNEKFRG SEQ ID NO: 31 CDR#2huAD208.14.1 V_(H) LVYPYIGGSSYNQQFKG SEQ ID NO: 41 CDR#2 hu139.10 V_(H)CDR#2 VIWAGGSTNYNSALMS SEQ ID NO: 51 muAD210.40.9 V_(H)DIYPGGGNTYYNEKLKG SEQ ID NO: 61 CDR#2 muAD209.9.1 V_(H)WINLYTGEPTFADDFKG SEQ ID NO: 71 CDR#2 huM25 V_(H) CDR#3 DRGNYRAWFGY SEQID NO: 12 huAD208.4.1 V_(H) GDNKYDAMDY SEQ ID NO: 22 CDR#3 huAD208.12.1V_(H) SDFGNYRWYFDV SEQ ID NO: 32 CDR#3 huAD208.14.1 V_(H) GDNNYDAMDY SEQID NO: 42 CDR#3 hu139.10 V_(H) CDR#3 HMITEDYYGMDY SEQ ID NO: 52muAD210.40.9 V_(H) WGDKKGNYFAY SEQ ID NO: 62 CDR#3 muAD209.9.1 V_(H)KGETYYRYDGFAY SEQ ID NO: 72 CDR#3

In some embodiments, an antibody and/or binding fragment composing ananti-huLRRC15 ADC comprises a V_(L) chain having three CDRs in whichV_(L) CDR#1, V_(L) CDR#2 and V_(L) CDR#3 have sequences selected fromtheir respective V_(L) CDR sequences in the table below:

CDR Sequence (N→C) Identifier huM25 V_(L) CDR#1 RASQDISNYLN SEQ ID NO:13 huAD208.4.1 V_(L) RASQSVSTSSYSYMH SEQ ID NO: 23 CDR#1 huAD208.12.1V_(L) RASQSSSNNLH SEQ ID NO: 33 CDR#1 huAD208.14.1 V_(L) RASQSVSTSTYNYMHSEQ ID NO: 43 CDR#1 hu139.10 V_(L) CDR#1 KSSQSLLNSRTRKNYLA SEQ ID NO: 53muAD210.40.9 V_(L) TASSSVYSSYLH SEQ ID NO: 63 CDR#1 muAD209.9.1 V_(L)RSSKSLLHSNGNTHLY SEQ ID NO: 73 CDR#1 huM25 V_(L) CDR#2 YTSRLHS SEQ IDNO: 14 huAD208.4.1 V_(L) YASSLES SEQ ID NO: 24 CDR#2 huAD208.12.1 V_(L)YVSQSIS SEQ ID NO: 34 CDR#2 huAD208.14.1 V_(L) YASNLES SEQ ID NO: 44CDR#2 hu139.10 V_(L) CDR#2 WASTRES SEQ ID NO: 54 muAD210.40.9 V_(L)STSNLAS SEQ ID NO: 64 CDR#2 muAD209.9.1 V_(L) RMSNLAS SEQ ID NO: 74CDR#2 huM25 V_(L) CDR#3 QQGEALPWT SEQ ID NO: 15 huAD208.4.1 V_(L)EQSWEIRT SEQ ID NO: 25 CDR#3 huAD208.12.1 V_(L) QQSNSWPFT SEQ ID NO: 35CDR#3 huAD208.14.1 V_(L) HHTWEIRT SEQ ID NO: 45 CDR#3 hu139.10 V_(L)CDR#3 KQSYNLPT SEQ ID NO: 55 muAD210.40.9 V_(L) HQYHRSPT SEQ ID NO: 65CDR#3 muAD209.9.1 V_(L) MQLLEYPYT SEQ ID NO: 75 CDR#3

In some embodiments, an antibody and/or binding fragment composing ananti-huLRRC15 ADC comprises a V_(H) chain having three CDRs in whichV_(H) CDR#1, V_(H) CDR#2 and V_(H) CDR#3 have sequences selected fromtheir respective V_(H) CDR sequences in the table below, and a V_(L)chain having three CDRs in which V_(L) CDR#1, V_(L) CDR#2 and V_(L)CDR#3 have sequences selected from their respective V_(L) CDR sequencesin the table below:

CDR Sequence (N→C) Identifier huM25 V_(H) CDR#1 SYWIE SEQ ID NO: 10huAD208.4.1 V_(H) DYYIH SEQ ID NO: 20 CDR#1 huAD208.12.1 V_(H) NYWMH SEQID NO: 30 CDR#1 huAD208.14.1 V_(H) DYYIH SEQ ID NO: 40 CDR#1 hu139.10V_(H) CDR#1 SYGVH SEQ ID NO: 50 muAD210.40.9 V_(H) NYWLG SEQ ID NO: 60CDR#1 muAD209.9.1 V_(H) NFGMN SEQ ID NO: 70 CDR#1 huM25 V_(H) CDR#2EILPGSDTTNYNEKFKD SEQ ID NO: 11 huAD208.4.1 V_(H) LVYPYIGGTNYNQKFKG SEQID NO: 21 CDR#2 huAD208.12.1 V_(H) MIHPNSGSTKHNEKFRG SEQ ID NO: 31 CDR#2huAD208.14.1 V_(H) LVYPYIGGSSYNQQFKG SEQ ID NO: 41 CDR#2 hu139.10 V_(H)CDR#2 VIWAGGSTNYNSALMS SEQ ID NO: 51 muAD210.40.9 V_(H)DIYPGGGNTYYNEKLKG SEQ ID NO: 61 CDR#2 muAD209.9.1 V_(H)WINLYTGEPTFADDFKG SEQ ID NO: 71 CDR#2 huM25 V_(H) CDR#3 DRGNYRAWFGY SEQID NO: 12 huAD208.4.1 V_(H) GDNKYDAMDY SEQ ID NO: 22 CDR#3 huAD208.12.1V_(H) SDFGNYRWYFDV SEQ ID NO: 32 CDR#3 huAD208.14.1 V_(H) GDNNYDAMDY SEQID NO: 42 CDR#3 hu139.10 V_(H) CDR#3 HMITEDYYGMDY SEQ ID NO: 52muAD210.40.9 V_(H) WGDKKGNYFAY SEQ ID NO: 62 CDR#3 muAD209.9.1 V_(H)KGETYYRYDGFAY SEQ ID NO: 72 CDR#3 huM25 V_(L) CDR#1 RASQDISNYLN SEQ IDNO: 13 huAD208.4.1 V_(L) RASQSVSTSSYSYMH SEQ ID NO: 23 CDR#1huAD208.12.1 V_(L) RASQSSSNNLH SEQ ID NO: 33 CDR#1 huAD208.14.1 V_(L)RASQSVSTSTYNYMH SEQ ID NO: 43 CDR#1 hu139.10 V_(L) CDR#1KSSQSLLNSRTRKNYLA SEQ ID NO: 53 muAD210.40.9 V_(L) TASSSVYSSYLH SEQ IDNO: 63 CDR#1 muAD209.9.1 V_(L) RSSKSLLHSNGNTHLY SEQ ID NO: 73 CDR#1huM25 V_(L) CDR#2 YTSRLHS SEQ ID NO: 14 huAD208.4.1 V_(L) YASSLES SEQ IDNO: 24 CDR#2 huAD208.12.1 V_(L) YVSQSIS SEQ ID NO: 34 CDR#2 huAD208.14.1V_(L) YASNLES SEQ ID NO: 44 CDR#2 hu139.10 V_(L) CDR#2 WASTRES SEQ IDNO: 54 muAD210.40.9 V_(L) STSNLAS SEQ ID NO: 64 CDR#2 muAD209.9.1 V_(L)RMSNLAS SEQ ID NO: 74 CDR#2 huM25 V_(L) CDR#3 QQGEALPWT SEQ ID NO: 15huAD208.4.1 V_(L) EQSWEIRT SEQ ID NO: 25 CDR#3 huAD208.12.1 V_(L)QQSNSWPFT SEQ ID NO: 35 CDR#3 huAD208.14.1 V_(L) HHTWEIRT SEQ ID NO: 45CDR#3 hu139.10 V_(L) CDR#3 KQSYNLPT SEQ ID NO: 55 muAD210.40.9 V_(L)HQYHRSPT SEQ ID NO: 65 CDR#3 muAD209.9.1 V_(L) MQLLEYPYT SEQ ID NO: 75CDR#3

In some embodiments, an antibody and/or binding fragment composing ananti-huLRRC15 ADC comprises a V_(H) chain having three CDRs in which:

V_(H) CDR#1 corresponds in sequence to SEQ ID NO:10, V_(H) CDR#2corresponds in sequence to SEQ ID NO:11 and V_(H) CDR#3 corresponds insequence to SEQ ID NO:12; or

V_(H) CDR#1 corresponds in sequence to SEQ ID NO:20, V_(H) CDR#2corresponds in sequence to SEQ ID NO:21 and V_(H) CDR#3 corresponds insequence to SEQ ID NO:22; or

V_(H) CDR#1 corresponds in sequence to SEQ ID NO:30, V_(H) CDR#2corresponds in sequence to SEQ ID NO:31 and V_(H) CDR#3 corresponds insequence to SEQ ID NO:32; or

V_(H) CDR#1 corresponds in sequence to SEQ ID NO:40, V_(H) CDR#2corresponds in sequence to SEQ ID NO:41 and V_(H) CDR#3 corresponds insequence to SEQ ID NO:42; or

V_(H) CDR#1 corresponds in sequence to SEQ ID NO:50, V_(H) CDR#2corresponds in sequence to SEQ ID NO:51 and V_(H) CDR#3 corresponds insequence to SEQ ID NO:52; or

V_(H) CDR#1 corresponds in sequence to SEQ ID NO:60, V_(H) CDR#2corresponds in sequence to SEQ ID NO:61 and V_(H) CDR#3 corresponds insequence to SEQ ID NO:62; or

V_(H) CDR#1 corresponds in sequence to SEQ ID NO:70, V_(H) CDR#2corresponds in sequence to SEQ ID NO:71 and V_(H) CDR#3 corresponds insequence to SEQ ID NO:72.

In some embodiments, an antibody and/or binding fragment composing ananti-huLRRC15 ADC comprises a V_(L) chain having three CDRs in which:

V_(L) CDR#1 corresponds in sequence to SEQ ID NO:13, V_(L) CDR#2corresponds in sequence to SEQ ID NO:14 and V_(L) CDR#3 corresponds insequence to SEQ ID NO:15; or

V_(L) CDR#1 corresponds in sequence to SEQ ID NO:23, V_(L) CDR#2corresponds in sequence to SEQ ID NO:24 and V_(L) CDR#3 corresponds insequence to SEQ ID NO:25; or

V_(L) CDR#1 corresponds in sequence to SEQ ID NO:33, V_(L) CDR#2corresponds in sequence to SEQ ID NO:34 and V_(L) CDR#3 corresponds insequence to SEQ ID NO:35; or

V_(L) CDR#1 corresponds in sequence to SEQ ID NO:43, V_(L) CDR#2corresponds in sequence to SEQ ID NO:44 and V_(L) CDR#3 corresponds insequence to SEQ ID NO:45; or

V_(L) CDR#1 corresponds in sequence to SEQ ID NO:53, V_(L) CDR#2corresponds in sequence to SEQ ID NO:54 and V_(L) CDR#3 corresponds insequence to SEQ ID NO:55; or

V_(L) CDR#1 corresponds in sequence to SEQ ID NO:63, V_(L) CDR#2corresponds in sequence to SEQ ID NO:64 and V_(L) CDR#3 corresponds insequence to SEQ ID NO:65; or

V_(L) CDR#1 corresponds in sequence to SEQ ID NO:73, V_(L) CDR#2corresponds in sequence to SEQ ID NO:74 and V_(L) CDR#3 corresponds insequence to SEQ ID NO:75.

In some embodiments, an antibody and/or binding fragment composing ananti-huLRRC15 ADC comprises:

a V_(H) chain having three CDRs in which V_(H) CDR#1 corresponds insequence to SEQ ID NO:10, V_(H) CDR#2 corresponds in sequence to SEQ IDNO:11 and V_(H) CDR#3 corresponds in sequence to SEQ ID NO:12 and aV_(L) chain having three CDRs in which V_(L) CDR#1 corresponds insequence to SEQ ID NO:13, V_(L) CDR#2 corresponds in sequence to SEQ IDNO:14 and V_(L) CDR#3 corresponds in sequence to SEQ ID NO:15; or

a V_(H) chain having three CDRs in which V_(H) CDR#1 corresponds insequence to SEQ ID NO:20, V_(H) CDR#2 corresponds in sequence to SEQ IDNO:21 and V_(H) CDR#3 corresponds in sequence to SEQ ID NO:22 and aV_(L) chain having three CDRs in which V_(L) CDR#1 corresponds insequence to SEQ ID NO:23, V_(L) CDR#2 corresponds in sequence to SEQ IDNO:24 and V_(L) CDR#3 corresponds in sequence to SEQ ID NO:25; or

a V_(H) chain having three CDRs in which V_(H) CDR#1 corresponds insequence to SEQ ID NO:30, V_(H) CDR#2 corresponds in sequence to SEQ IDNO:31 and V_(H) CDR#3 corresponds in sequence to SEQ ID NO:32 and aV_(L) chain having three CDRs in which V_(L) CDR#1 corresponds insequence to SEQ ID NO:33, V_(L) CDR#2 corresponds in sequence to SEQ IDNO:34 and V_(L) CDR#3 corresponds in sequence to SEQ ID NO:35; or

a V_(H) chain having three CDRs in which V_(H) CDR#1 corresponds insequence to SEQ ID NO:40, V_(H) CDR#2 corresponds in sequence to SEQ IDNO:41 and V_(H) CDR#3 corresponds in sequence to SEQ ID NO:42 and aV_(L) chain having three CDRs in which V_(L) CDR#1 corresponds insequence to SEQ ID NO:43, V_(L) CDR#2 corresponds in sequence to SEQ IDNO:44 and V_(L) CDR#3 corresponds in sequence to SEQ ID NO:45; or

a V_(H) chain having three CDRs in which V_(H) CDR#1 corresponds insequence to SEQ ID NO:50, V_(H) CDR#2 corresponds in sequence to SEQ IDNO:51 and V_(H) CDR#3 corresponds in sequence to SEQ ID NO:52 and aV_(L) chain having three CDRs in which V_(L) CDR#1 corresponds insequence to SEQ ID NO:53, V_(L) CDR#2 corresponds in sequence to SEQ IDNO:54 and V_(L) CDR#3 corresponds in sequence to SEQ ID NO:55; or

a V_(H) chain having three CDRs in which V_(H) CDR#1 corresponds insequence to SEQ ID NO:60, V_(H) CDR#2 corresponds in sequence to SEQ IDNO:61 and V_(H) CDR#3 corresponds in sequence to SEQ ID NO:62 and aV_(L) chain having three CDRs in which V_(L) CDR#1 corresponds insequence to SEQ ID NO:63, V_(L) CDR#2 corresponds in sequence to SEQ IDNO:64 and V_(L) CDR#3 corresponds in sequence to SEQ ID NO:65; or

a V_(H) chain having three CDRs in which V_(H) CDR#1 corresponds insequence to SEQ ID NO:70, V_(H) CDR#2 corresponds in sequence to SEQ IDNO:71 and V_(H) CDR#3 corresponds in sequence to SEQ ID NO:72 and aV_(L) chain having three CDRs in which V_(L) CDR#1 corresponds insequence to SEQ ID NO:73, V_(L) CDR#2 corresponds in sequence to SEQ IDNO:74 and V_(L) CDR#3 corresponds in sequence to SEQ ID NO:75.

In some embodiments, an antibody and/or binding fragment composing ananti-huLRRC15 ADC comprises a V_(H) chain having a sequencecorresponding to a sequence selected from one of the sequences in thetable below:

Chain Sequence (N→C) Identifier huM25 V_(H)EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAP SEQ ID NO: 16GQGLEWIGEILPGSDTTNYNEKFKDRATFTSDTSINTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSS huAD208.4.1 V_(H)EVQLVQSGAEVKKPGSSVKVSCKASGFTFTDYYIHWVKQAP SEQ ID NO: 26GQGLEWIGLVYPYIGGTNYNQKFKGKATLTVDTSTTTAYMEMSSLRSEDTAVYYCARGDNKYDAMDYWGQGTTVTVSS huAD208.12.1 V_(H)EVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWMHWVKQA SEQ ID NO: 36PGQGLEWIGMIHPNSGSTKHNEKFRGKATLTVDESTTTAYMELSSLRSEDTAVYYCARSDFGNYRWYFDVWGQGTTVTVSS huAD208.14.1 V_(H)EVQLVQSGAEVKKPGSSVKVSCKASGFTFTDYYIHWVKQAP SEQ ID NO: 46GQGLEWIGLVYPYIGGSSYNQQFKGKATLTVDTSTSTAYMELSSLRSEDTAVYYCARGDNNYDAMDYWGQGTTVTVSS hu139.10 V_(H)EVQLVESGGGLVQPGGSLRLSCAVSGFSLTSYGVHWVRQAT SEQ ID NO: 56GKGLEWLGVIWAGGSTNYNSALMSRLTISKENAKSSVYLQMNSLRAGDTAMYYCATHMITEDYYGMDYWGQGTTVTVSS muAD210.40.9 V_(H)QVQLQQSGAELVRPGTSVKISCKASGYDFTNYWLGWVKQRP SEQ ID NO: 66GHGLEWIGDIYPGGGNTYYNEKLKGKATLTADKSSSTAYIHLISLTSEDSSVYFCARWGDKKGNYFAYWGQGTLVTVSA muAD209.9.1 V_(H)QIQLVQSGPELKKPGETVKISCKASGFAITNFGMNWVKQAPG SEQ ID NO: 76KGLKWMGWINLYTGEPTFADDFKGRFAFSLETSASTAYLQINNLKNEDTVIYFCARKGETYYRYDGFAYWGQGTLVTVSA

In some embodiments, an antibody and/or binding fragment composing ananti-huLRRC15 ADC comprises a V_(L) chain having a sequencecorresponding to a sequence selected from one of the sequences in thetable below:

Chain Sequence (N→C) Identifier huM25 V_(L)DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPG SEQ ID NO: 17GAVKFLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDF ATYFCQQGEALPWTFGGGTKVEIKhuAD208.4.1 V_(L) DIVLTQSPDSLAVSLGERATINCRASQSVSTSSYSYMHWYQ SEQ ID NO:27 QKPGQPPKLLIKYASSLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCEQSWEIRTFGGGTKVEIK huAD208.12.1 V_(L)EIVLTQSPATLSLSPGERATLSCRASQSSSNNLHWYQQKPG SEQ ID NO: 37QAPRVLIKYVSQSISGIPARFSGSGSGTDFTLTISSLEPEDFA VYFCQQSNSWPFTFGQGTKLEIKhuAD208.14.1 V_(L) DIVLTQSPDSLAVSLGERATISCRASQSVSTSTYNYMHWYQ SEQ ID NO:47 QKPGQPPKLLVKYASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHHTWEIRTFGGGTKVEIK hu139.10 V_(L)DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAW SEQ ID NO: 57YQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISS LQAEDVAVYYCKQSYNLPTFGGGTKVEIKmuAD210.40.9 V_(L) QIVLTQSPAIMSASLGERVTMTCTASSSVYSSYLHWYQQK SEQ ID NO:67 PGSSPKLWIYSTSNLASGVPGRFSGSGSGTSYSLTISSMEAE DAATYYCHQYHRSPTFGGGTKLEIKmuAD209.9.1 V_(L) DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTHLYWF SEQ ID NO:77 LQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQLLEYPYTFGGGTKLEIE

In some embodiments, an antibody and/or binding fragment composing ananti-huLRRC15 ADC comprises:

a V_(H) chain having a sequence corresponding to SEQ ID NO:16 and aV_(L) chain corresponding in sequence to SEQ ID NO:17; or

a V_(H) chain having a sequence corresponding to SEQ ID NO:26 and aV_(L) chain corresponding in sequence to SEQ ID NO:27; or

a V_(H) chain having a sequence corresponding to SEQ ID NO:36 and aV_(L) chain corresponding in sequence to SEQ ID NO:37; or

a V_(H) chain having a sequence corresponding to SEQ ID NO:46 and aV_(L) chain corresponding in sequence to SEQ ID NO:47; or

a V_(H) chain having a sequence corresponding to SEQ ID NO:56 and aV_(L) chain corresponding in sequence to SEQ ID NO:57; or

a V_(H) chain having a sequence corresponding to SEQ ID NO:66 and aV_(L) chain corresponding in sequence to SEQ ID NO:67; or

a V_(H) chain having a sequence corresponding to SEQ ID NO:76 and aV_(L) chain corresponding in sequence to SEQ ID NO:77.

The anti-huLRRC15 ADCs have myriad uses, and in particular are usefultherapeutically for the treatment of huLRRC15 stromal(+)/cancer(−)tumors in humans. Accordingly, in some embodiments, an anti-huLRRC15antibody and/or binding fragment composing an anti-huLRRC15 ADC issuitable for administration to humans. In a specific embodiment, theanti-huLRRC15 antibody is humanized. In some embodiments, the humanizedanti-huLRRC15 antibody and/or binding fragment composing ananti-huLRRC15 ADC is an antibody and/or binding fragment comprising aV_(H) chain corresponding in sequence to SEQ ID NO:16, SEQ ID NO:26, SEQID NO:36, SEQ ID NO:46 or SEQ ID NO:56 and a V_(L) chain correspondingin sequence to SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:47 orSEQ ID NO:57. In some embodiments, the humanized anti-huLRRC15 antibodyand/or binding fragment is a full length antibody selected from huM25,huM25-S239C, huAD208.4.1, huAD208.4.1-S239C, huAD208.12.1, huAD208.14.1and hu139.10.

In some embodiments, an anti-huLRRC15 antibody and/or binding fragmentcomposing an anti-huLRRC15 ADC is an IgG₁.

In some embodiments, an anti-huLRRC15 antibody composing ananti-huLRRC15 ADC comprises a heavy chain having a constant regioncorresponding in sequence to residues 121-450 of SEQ ID NO:18 by linearamino acid sequence numbering.

In some embodiments, an anti-huLRRC15 antibody composing ananti-huLRRC15 ADC comprises a light chain having a constant regioncorresponding in sequence to residues 108-214 of SEQ ID NO:19 by linearamino acid sequence numbering.

In some embodiments, an anti-huLRRC15 antibody composing ananti-huLRRC15 ADC comprises a heavy chain having a constant regioncorresponding in sequence to residues 121-450 of SEQ ID NO:18 and alight chain having a constant region corresponding in sequence toresidues 108-214 of SEQ ID NO:19 by linear amino acid sequencenumbering.

In some embodiments, anti-huLRRC15 antibodies and/or binding fragmentscomposing an anti-huLRRC15 ADC compete for binding huLRRC15 on cellsexpressing huLRRC15, or the sECD of huLRRC15, in in vitro assays with areference antibody. The reference antibody may be any antibody thatspecifically binds huLRRC15 within a region of the sECD. In one specificembodiment, the reference antibody is huM25, huM25-S239C, huAD208.4.1,huAD208.4.1-S239C, huAD208.12.1, huAD208.14.1, hu139.10, muAD210.40.9 ormuAD209.9.1.

In some embodiments, an anti-huLRRC15 antibody composing ananti-huLRRC15 ADC has a heavy chain amino acid sequence (N→C) accordingto:

(SEQ ID NO: 100) EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYNEKFKDRATFTSDTSINTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVIVHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K,

and a light chain amino acid sequence (N→C) according to SEQ ID NO: 19,wherein the underlined amino acids represent the CDRs and the italicizedamino acids represent the constant regions.

In some embodiments, an anti-huLRRC15 antibody composing ananti-huLRRC15 ADC has a heavy chain amino acid sequence (N→C) accordingto:

(SEQ ID NO: 102) EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYNEKFKDRATFTSDTSINTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVIVHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG,and a light chain amino acid sequence (N→C) according to SEQ ID NO: 19,wherein the underlined amino acids represent the CDRs and the italicizedamino acids represent the constant regions.

In some embodiments, an anti-huLRRC15 antibody composing ananti-huLRRC15 ADC has a heavy chain amino acid sequence (N→C) accordingto:

(SEQ ID NO: 103) EVQLVQSGAEVKKPGASVKVSCKASGYKFSSYWIEWVKQAPGQGLEWIGEILPGSDTTNYNEKFKDRATFTSDTSINTAYMELSRLRSDDTAVYYCARDRGNYRAWFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,and a light chain amino acid sequence (N→C) according to SEQ ID NO: 19,wherein the underlined amino acids represent the CDRs and the italicizedamino acids represent the constant regions.

In some embodiments, an anti-huLRRC15 antibody composing ananti-huLRRC15 ADC has a heavy chain amino acid sequence (N→C) accordingto SEQ ID NO: 18 or 102;

and a light chain amino acid sequence (N→C) according to:

(SEQ ID NO: 110) DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGAVKFLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGEALPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEA,wherein the underlined amino acids represent the CDRs and the italicizedamino acids represent the constant regions.

In some embodiments, an anti-huLRRC15 antibody composing ananti-huLRRC15 ADC has a heavy chain amino acid sequence (N→C) accordingto:

(SEQ ID NO: 28) EVQLVQSGAEVKKPGSSVKVSCKASGFTFTDYYIHWVKQAPGQGLEWIGLVYPYIGGTNYNQKFKGKATLTVDTSTTTAYMEMSSLRSEDTAVYYCARGDNKYDAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;and a light chain amino acid sequence (N→C) according to:

(SEQ ID NO: 29) DIVLTQSPDSLAVSLGERATINCRASQSVSTSSYSYMHWYQQKPGQPPKLLIKYASSLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCEQSWEIRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC,wherein the underlined amino acids represent the CDRs and the italicizedamino acids represent the constant regions.

In some embodiments, an anti-huLRRC15 antibody composing ananti-huLRRC15 ADC has a heavy chain amino acid sequence (N→C) accordingto:

(SEQ ID NO: 101) EVQLVQSGAEVKKPGSSVKVSCKASGFTFTDYYIHWVKQAPGQGLEWIGLVYPYIGGTNYNQKFKGKATLTVDTSTTTAYMEMSSLRSEDTAVYYCARGDNKYDAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;and a light chain amino acid sequence (N→C) according to SEQ ID NO: 29,wherein the underlined amino acids represent the CDRs and the italicizedamino acids represent the constant regions.

In some embodiments, an anti-huLRRC15 antibody composing ananti-huLRRC15 ADC has a heavy chain amino acid sequence (N→C) accordingto:

(SEQ ID NO: 104) EVQLVQSGAEVKKPGSSVKVSCKASGFTFTDYYIHWVKQAPGQGLEWIGLVYPYIGGTNYNQKFKGKATLTVDTSTTTAYMEMSSLRSEDTAVYYCARGDNKYDAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG;and a light chain amino acid sequence (N→C) according to SEQ ID NO: 29,wherein the underlined amino acids represent the CDRs and the italicizedamino acids represent the constant regions.

In some embodiments, an anti-huLRRC15 antibody composing ananti-huLRRC15 ADC has a heavy chain amino acid sequence (N→C) accordingto:

(SEQ ID NO: 105) EVQLVQSGAEVKKPGSSVKVSCKASGFTFTDYYIHWVKQAPGQGLEWIGLVYPYIGGTNYNQKFKGKATLTVDTSTTTAYMEMSSLRSEDTAVYYCARGDNKYDAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG;and a light chain amino acid sequence (N→C) according to SEQ ID NO: 29,wherein the underlined amino acids represent the CDRs and the italicizedamino acids represent the constant regions.

In some embodiments, an anti-huLRRC15 antibody composing ananti-huLRRC15 ADC has a heavy chain amino acid sequence (N→C) accordingto SEQ ID NO: 28 or 104;

and a light chain amino acid sequence (N→C) according to:

(SEQ ID NO: 111) DIVLTQSPDSLAVSLGERATINCRASQSVSTSSYSYMHWYQQKPGQPPKLLIKYASSLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCEQSWEIRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEA,wherein the underlined amino acids represent the CDRs and the italicizedamino acids represent the constant regions.

Assays for competition include, but are not limited to, a radioactivematerial labeled immunoassay (RIA), an enzyme-linked immunosorbent assay(ELISA), a sandwich ELISA, flow cytometry assays and surface plasmonresonance assays.

In one exemplary embodiment of conducting an antibody competition assaybetween a reference antibody and a test antibody (irrespective ofspecies or isotype), one may first label the reference with a detectablelabel, such as a fluorophore, biotin or an enzymatic (or evenradioactive label) to enable subsequent detection. In this case, cellsexpressing huLRRC15, or the sECD of huLRRC15, are incubated withunlabeled test antibody, labeled reference antibody is added, and theintensity of the bound label is measured. If the test antibody competeswith the labeled reference antibody by binding to the same, proximal oroverlapping epitope, the intensity of the detection signal will bedecreased relative to a control reaction carried out without testantibody.

In a specific embodiment of this assay, the concentration of labeledreference antibody that yields 80% of maximal binding (“conc_(80%)”)under the assay conditions (e.g., a specified density of cells or aspecified concentration of sECD) is first determined, and a competitionassay is carried out with 10× conc_(80%) of unlabeled test antibody andconc_(80%) of labeled reference antibody.

In another exemplary embodiment of conducting a flow cytometrycompetition assay, cells expressing huLRRC15 are incubated with atitration series of antibodies comprising increasing rations ofunlabeled test antibody versus fluorescently labeled anti-huLRRC15reference antibody. The labeled reference anti-huLRRC15 antibody is usedat a fixed concentration X (for example, X=1 μg/ml) and the unlabeledtest antibody is used in a range of concentrations (for example, from10′X to 100X). Cells or sECD is incubated with both unlabeled testantibody and labeled reference antibody concurrently. Flow cytometrydata is normalized relative to fluorescently labeled reference antibodyalone, where the fluorescence intensity of a sample carried out withoutunlabeled test antibody is assigned 100% binding. If a test antibodycompetes for binding huLRRC15 with the labeled reference antibody, anassay carried out with equal concentration of each (for example, 1 μg/mLof unlabeled test antibody and 1 μg/mL of labeled reference antibody)will yield an approx. 50% reduction in fluorescence intensity ascompared to the 100% control, indicating approx. 50% binding.

The inhibition can be expressed as an inhibition constant, or K_(i),which is calculated according to the following formula:K _(i)=IC₅₀/(1+[reference Ab concentration]/K _(d)),where IC₅₀ is the concentration of test antibody that yields a 50%reduction in binding of the reference antibody and K_(d) is thedissociation constant of the reference antibody, a measure of itsaffinity for huLRRC15. Antibodies that compete with referenceanti-huLRRC15 antibodies can have a K_(i) from 10 pM to 10 nM underassay conditions described herein.

In various embodiments, a test antibody is considered to compete with areference antibody if it decreases binding of the reference antibody tocells expressing huLRRC15 or the sECD by at least about 20% or more, forexample, by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%or even more, or by a percentage ranging between any of the foregoingvalues, at a reference antibody concentration that is 80% of maximalbinding under the specific assay conditions used, and a test antibodyconcentration that is 10-fold higher than the reference antibodyconcentration.

In various embodiments of a flow cytometry competition assay, a testantibody is considered to compete with a reference antibody if itdecreases binding of the reference antibody to cells expressing huLRRC15by at least about 20% or more, for example, by at least about 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95% or even more, or by a percentageranging between any of the foregoing values, at a concentration of testantibody that is 10× greater than that of the reference antibody.

A specific assay and assay conditions useful for assessing whether anantibody competes for binding huLRRC15 with a reference antibody asdescribed herein is provided in Example 3.

The anti-huLRRC15 antibodies described herein may be used in the non-ADCcontext for a variety of purposes, such as to assist purification ofhuLRRC15 and/or huLRRC15 sECD, in vitro, in vivo and ex vivodiagnostics, cell and/or tissue stains, etc. As a specific example, theantibodies have use in immunoassays for qualitatively and/orquantitatively measuring levels of huLRRC15 in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, Second Edition(Cold Spring Harbor Laboratory Press, 1988).

For such uses, detection can be facilitated by coupling the antibody toa detectable substance. Examples of detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive materials, positronemitting metals using various positron emission tomographies, andnonradioactive paramagnetic metal ions. The detectable substance can becoupled or conjugated either directly to the antibody (or fragmentthereof) or indirectly, through an intermediate (such as, for example, alinker known in the art) using techniques known in the art. Examples ofenzymatic labels include luciferases (e.g., firefly luciferase andbacterial luciferase; U.S. Pat. No. 4,737,456), luciferin,2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidasesuch as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, acetylcholinesterase, glucoamylase, lysozyme,saccharide oxidases (e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase), heterocyclic oxidases (such asuricase and xanthine oxidase), lactoperoxidase, microperoxidase, and thelike. Examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In or ^(99m)Tc.

Detection of expression of huLRRC15 generally involves contacting abiological sample (cells, tissue, or body fluid of an individual) withone or more anti-huLRRC15 antibodies described herein (optionallyconjugated to detectable moiety), and detecting whether or not thesample is positive for huLRRC15 expression, or whether the sample hasaltered (e.g., reduced or increased) expression as compared to a controlsample.

7.3.2. Polynucleotides Encoding Anti-huLRRC15 Antibodies, ExpressionSystems and Methods of Making the Antibodies

Anti-huLRRC15 antibodies can be prepared by recombinant expression ofimmunoglobulin light and heavy chain genes in a host cell. To express anantibody recombinantly, a host cell is transfected with one or morerecombinant expression vectors carrying DNA fragments encoding theimmunoglobulin light and heavy chains of the antibody such that thelight and heavy chains are expressed in the host cell and, optionally,secreted into the medium in which the host cells are cultured, fromwhich medium the antibodies can be recovered. Standard recombinant DNAmethodologies are used to obtain antibody heavy and light chain genes,incorporate these genes into recombinant expression vectors andintroduce the vectors into host cells, such as those described inMolecular Cloning; A Laboratory Manual, Second Edition (Sambrook,Fritsch and Maniatis (eds), Cold Spring Harbor, N.Y., 1989), CurrentProtocols in Molecular Biology (Ausubel, F. M. et al., eds., GreenePublishing Associates, 1989) and in U.S. Pat. No. 4,816,397.

To generate nucleic acids encoding such anti-huLRRC15 antibodies, DNAfragments encoding the light and heavy chain variable regions are firstobtained. These DNAs can be obtained by amplification and modificationof germline DNA or cDNA encoding light and heavy chain variablesequences, for example using the polymerase chain reaction (PCR).Germline DNA sequences for human heavy and light chain variable regiongenes are known in the art (See, e.g., the “VBASE” human germlinesequence database; see also Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson et al., 1992, J.Mol. Biol. 22T:116-198; and Cox et al., 1994, Eur. J. Immunol.24:827-836; the contents of each of which are incorporated herein byreference).

Once DNA fragments encoding anti-huLRRC15 antibody-related V_(H) andV_(L) segments are obtained, these DNA fragments can be furthermanipulated by standard recombinant DNA techniques, for example toconvert the variable region genes to full-length antibody chain genes,to Fab fragment genes or to a scFv gene. In these manipulations, aV_(L)- or V_(H)-encoding DNA fragment is operatively linked to anotherDNA fragment encoding another protein, such as an antibody constantregion or a flexible linker. The term “operatively linked,” as used inthis context, is intended to mean that the two DNA fragments are joinedsuch that the amino acid sequences encoded by the two DNA fragmentsremain in-frame.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CH₁,CH₂, CH₃ and, optionally, CH₄). The sequences of human heavy chainconstant region genes are known in the art (See, e.g., Kabat et al.,1991, Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242) and DNA fragments encompassing these regions can be obtained bystandard PCR amplification. The heavy chain constant region can be anIgG₁, IgG₂, IgG₃, IgG₄, IgA, IgE, IgM or IgD constant region, but incertain embodiments is an IgG₁ or IgG₄ constant region. For a Fabfragment heavy chain gene, the V_(H)-encoding DNA can be operativelylinked to another DNA molecule encoding only the heavy chain CH₁constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, CL. The sequences of humanlight chain constant region genes are known in the art (See, e.g., Kabatet al., 1991, Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region, but in certain embodiments isa kappa constant region. To create a scFv gene, the V_(H)- andV_(L)-encoding DNA fragments are operatively linked to another fragmentencoding a flexible linker, e.g., encoding the amino acid sequence(Gly₄˜Ser)₃ (SEQ ID NO:82), such that the V_(H) and V_(L) sequences canbe expressed as a contiguous single-chain protein, with the V_(L) andV_(H) regions joined by the flexible linker (See, e.g., Bird et al.,1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci.USA 85:5879-5883; McCafferty et al., 1990, Nature 348:552-554).

To express the anti-huLRRC15 antibodies, DNAs encoding partial orfull-length light and heavy chains, obtained as described above, areinserted into expression vectors such that the genes are operativelylinked to transcriptional and translational control sequences. In thiscontext, the term “operatively linked” is intended to mean that anantibody gene is ligated into a vector such that transcriptional andtranslational control sequences within the vector serve their intendedfunction of regulating the transcription and translation of the antibodygene. The expression vector and expression control sequences are chosento be compatible with the expression host cell used. The antibody lightchain gene and the antibody heavy chain gene can be inserted intoseparate vectors or, more typically, both genes are inserted into thesame expression vector.

The antibody genes are inserted into the expression vector by standardmethods (e.g., ligation of complementary restriction sites on theantibody gene fragment and vector, or blunt end ligation if norestriction sites are present). Prior to insertion of the anti-huLRRC15antibody-related light or heavy chain sequences, the expression vectorcan already carry antibody constant region sequences. For example, oneapproach to converting the anti-hPG monoclonal antibody-related V_(H)and V_(L) sequences to full-length antibody genes is to insert them intoexpression vectors already encoding heavy chain constant and light chainconstant regions, respectively, such that the V_(H) segment isoperatively linked to the CH segment(s) within the vector and the V_(L)segment is operatively linked to the CL segment within the vector.Additionally or alternatively, the recombinant expression vector canencode a signal peptide that facilitates secretion of the antibody chainfrom a host cell. The antibody chain gene can be cloned into the vectorsuch that the signal peptide is linked in-frame to the amino terminus ofthe antibody chain gene. The signal peptide can be an immunoglobulinsignal peptide or a heterologous signal peptide (i.e., a signal peptidefrom a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors carry regulatory sequences that control the expression of theantibody chain genes in a host cell. The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals) that control the transcriptionor translation of the antibody chain genes. Such regulatory sequencesare described, for example, in Goeddel, Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif., 1990. Itwill be appreciated by those skilled in the art that the design of theexpression vector, including the selection of regulatory sequences maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Suitable regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from cytomegalovirus (CMV) (such asthe CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40promoter/enhancer), adenovirus, (e.g., the adenovirus major latepromoter (AdMLP)) and polyoma. For further description of viralregulatory elements, and sequences thereof, see, e.g., U.S. Pat. No.5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al., and U.S.Pat. No. 4,968,615 by Schaffner et al.

Recombinant expression vectors of the disclosure can carry sequences inaddition to the antibody chain genes and regulatory sequences, such assequences that regulate replication of the vector in host cells (e.g.,origins of replication) and selectable marker genes. Selectable markergenes facilitate selection of host cells into which the vector has beenintroduced (See, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and5,179,017, all by Axel et al.). For example, typically a selectablemarker gene confers resistance to drugs, such as G418, hygromycin ormethotrexate, on a host cell into which the vector has been introduced.Suitable selectable marker genes include the dihydrofolate reductase(DHFR) gene (for use in DHFR⁻ host cells with methotrexateselection/amplification) and the neo gene (for G418 selection). Forexpression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, lipofection, calcium-phosphateprecipitation, DEAE—dextran transfection and the like.

It is possible to express anti-huLRRC15 antibodies composinganti-huLRRC15 ADCs in either prokaryotic or eukaryotic host cells. Incertain embodiments, expression of antibodies is performed in eukaryoticcells, e.g., mammalian host cells, of optimal secretion of a properlyfolded and immunologically active antibody. Exemplary mammalian hostcells for expressing the recombinant antibodies of the disclosureinclude Chinese Hamster Ovary (CHO cells) (including DHFR⁻ CHO cells,described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA77:4216-4220, used with a DHFR selectable marker, e.g., as described inKaufman and Sharp, 1982, Mol. Biol. 159:601-621), NSO myeloma cells, COScells and SP2 cells. When recombinant expression vectors encodingantibody genes are introduced into mammalian host cells, the antibodiesare produced by culturing the host cells for a period of time sufficientto allow for expression of the antibody in the host cells or secretionof the antibody into the culture medium in which the host cells aregrown. Antibodies can be recovered from the culture medium usingstandard protein purification methods. Host cells can also be used toproduce portions of intact antibodies, such as Fab fragments or scFvmolecules. It is understood that variations on the above procedure arewithin the scope of the present disclosure. For example, it can bedesirable to transfect a host cell with DNA encoding either the lightchain or the heavy chain (but not both) of an anti-huLRRC15 antibody.

Recombinant DNA technology can also be used to remove some or all of theDNA encoding either or both of the light and heavy chains that is notnecessary for binding to huLRRC15. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies of thedisclosure.

For recombinant expression of an anti-huLRRC15 antibody, the host cellcan be co-transfected with two expression vectors, the first vectorencoding a heavy chain derived polypeptide and the second vectorencoding a light chain derived polypeptide. The two vectors can containidentical selectable markers, or they can each contain a separateselectable marker. Alternatively, a single vector can be used whichencodes both heavy and light chain polypeptides.

Once a nucleic acid encoding one or more portions of an anti-huLRRC15antibody is obtained, further alterations or mutations can be introducedinto the coding sequence, for example to generate nucleic acids encodingantibodies with different CDR sequences, antibodies with reducedaffinity to the Fc receptor, or antibodies of different subclasses.

Antibodies and/or binding fragments composing anti-huLRRC15 ADCs canalso be produced by chemical synthesis (e.g., by the methods describedin Solid Phase Peptide Synthesis, 2^(nd) ed., 1984 The Pierce ChemicalCo., Rockford, Ill.). Variant antibodies can also be generated using acell-free platform, See, e.g., Chu et al., Biochemia No. 2, 2001 (RocheMolecular Biologicals) and Murray et al., 2013, Current Opinion inChemical Biology, 17:420-426.

Once an anti-huLRRC15 antibody and/or binding fragment has been producedby recombinant expression, it can be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theanti-huLRRC15 antibodies and/or binding fragments can be fused toheterologous polypeptide sequences described herein or otherwise knownin the art to facilitate purification.

Once isolated, the anti-huLRRC15 antibody and/or binding fragment can,if desired, be further purified, e.g., by high performance liquidchromatography. (see, e.g., Fisher, Laboratory Techniques InBiochemistry And Molecular Biology, Work and Burdon, eds., Elsevier,1980), or by gel filtration chromatography on a Superdex™ 75 column(Pharmacia Biotech AB, Uppsala, Sweden).

7.4. Specific Anti-huLRRC15 Antibody Drug Conjugates

As mentioned, anti-huLRRC15 ADCs generally comprise an anti-huLRRC15antigen binding moiety, such as an anti-huLRRC15 antibody and/or bindingfragment, having one or more cytotoxic and/or cytostatic agents, whichmay be the same or different, linked thereto by way of one or morelinkers, which may also be the same or different. In specificembodiments, the anti-huLRRC15 ADCs are compounds according tostructural formula (I):[D-L-XY]_(n)-Ab  (I)

or salts thereof, where each “D” represents, independently of theothers, a cytotoxic and/or cytostatic agent (“drug”); each “L”represents, independently of the others, a linker; “Ab” represents ananti-huLRRC15 antigen binding moiety, such as an anti-huLRRC15 antibodyor binding fragment; each “XY” represents a linkage formed between afunctional group R^(x) on the linker and a “complementary” functionalgroup W on the antigen binding moiety; and n represents the number ofdrugs linked to Ab, or the drug-to-antibody ratio (DAR), of the ADC.

Specific embodiments of various antibodies or binding fragments (Ab)that may compose ADCs according to structural formula (I) include thevarious embodiments of anti-huLRRC15 antibodies and/or binding fragmentsdescribed above.

In some specific embodiments of the ADCs or salts of structural formula(I), each D is the same and/or each L is the same.

Specific embodiments of cytotoxic and/or cytostatic agents (D) andlinkers (L) that may compose the anti-huLRRC15 ADCs, as well as thenumber of cytotoxic and/or cytostatic agents linked to the anti-huLRRC15ADCs, are described in more detail below.

7.4.1. Cytotoxic and/or Cytostatic Agents

The cytotoxic and/or cytostatic agents may be any agents known toinhibit the growth and/or replication of and/or kill cells, and inparticular cancer and/or tumor cells. Numerous agents having cytotoxicand/or cytostatic properties are known in the literature. Non-limitingexamples of classes of cytotoxic and/or cytostatic agents include, byway of example and not limitation, radionuclides, alkylating agents, DNAcross-linking agents, DNA intercalating agents (e.g., groove bindingagents such as minor groove binders), cell cycle modulators, apoptosisregulators, kinase inhibitors, protein synthesis inhibitors,mitochondria inhibitors, nuclear export inhibitors, topoisomerase Iinhibitors, topoisomerase II inhibitors, RNA/DNA antimetabolites andantimitotic agents.

Specific non-limiting examples of agents within certain of these variousclasses are provided below.

Alkylating Agents:

asaley (L-Leucine,N—[N-acetyl-4-[bis-(2-chloroethyl)amino]-DL-phenylalanyl]-, ethylester);AZQ (1,4-cyclohexadiene-1,4-dicarbamic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo-, diethyl ester); BCNU(N,N′-Bis(2-chloroethyl)-N-nitrosourea); busulfan (1,4-butanedioldimethanesulfonate); (carboxyphthalato)platinum; CBDCA(cis-(1,1-cyclobutanedicarboxylato)diammineplatinum(II))); CCNU(N-(2-chloroethyl)-N′-cyclohexyl-N-nitrosourea); CHIP (iproplatin; NSC256927); chlorambucil; chlorozotocin (2-[[[(2-chloroethyl)nitrosoamino]carbonyl]amino]-2-deoxy-D-glucopyranose); cis-platinum(cisplatin); clomesone; cyanomorpholinodoxorubicin; cyclodisone;dianhydrogalactitol (5,6-diepoxydulcitol); fluorodopan((5-[(2-chloroethyl)-(2-fluoroethyl)amino]-6-methyl-uracil); hepsulfam;hycanthone; indolinobenzodiazepine dimer DGN462; melphalan; methyl CCNU((1-(2-chloroethyl)-3-(trans-4-methylcyclohexane)-1-nitrosourea);mitomycin C; mitozolamide; nitrogen mustard ((bis(2-chloroethyl)methylamine hydrochloride); PCNU((1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea));piperazine alkylator ((1-(2-chloroethyl)-4-(3-chloropropyl)-piperazinedihydrochloride)); piperazinedione; pipobroman(N,N′-bis(3-bromopropionyl) piperazine); porfiromycin (N-methylmitomycinC); spirohydantoin mustard; teroxirone (triglycidylisocyanurate);tetraplatin; thio-tepa (N,N′,N″-tri-1,2-ethanediylthio phosphoramide);triethylenemelamine; uracil nitrogen mustard (desmethyldopan); Yoshi-864((bis(3-mesyloxy propyl)amine hydrochloride).

DNA Alkylating-Like Agents:

Cisplatin; Carboplatin; Nedaplatin; Oxaliplatin; Satraplatin; Triplatintetranitrate; Procarbazine; altretamine; dacarbazine; mitozolomide;temozolomide.

Alkylating Antineoplastic Agents:

Carboquone; Carmustine; Chlornaphazine; Chlorozotocin; Duocarmycin;Evofosfamide; Fotemustine; Glufosfamide; Lomustine; Mannosulfan;Nimustine; Phenanthriplatin; Pipobroman; Ranimustine; Semustine;Streptozotocin; ThioTEPA; Treosulfan; Triaziquone; Triethylenemelamine;Triplatin tetranitrate.

DNA Replication and Repair Inhibitors:

Altretamine; Bleomycin; Dacarbazine; Dactinomycin; Mitobronitol;Mitomycin; Pingyangmycin; Plicamycin; Procarbazine; Temozolomide;ABT-888 (veliparib); olaparib; KU-59436; AZD-2281; AG-014699; BSI-201;BGP-15; INO-1001; ONO-2231.

Cell Cycle Modulators:

Paclitaxel; Nab-Paclitaxel; Docetaxel; Vincristine; Vinblastine;ABT-348; AZD-1152; MLN-8054; VX-680; Aurora A-specific kinaseinhibitors; Aurora B-specific kinase inhibitors and pan-Aurora kinaseinhibitors; AZD-5438; BMI-1040; BMS-032; BMS-387; CVT-2584;flavopyridol; GPC-286199; MCS-5A; PD0332991; PHA-690509; seliciclib(CYC-202, R-roscovitine); ZK-304709; AZD4877, ARRY-520: GSK923295A.

Apoptosis Regulators:

AT-101 ((−)gossypol); G3139 or oblimersen (Bcl-2-targeting antisenseoligonucleotide); IPI-194; IPI-565;N-(4-(4-((4′-chloro(1,1′-biphenyl)-2-yl)methyl)piperazin-1-ylbenzoyl)-4-(((1R)-3-(dimethylamino)-1-((phenylsulfanyl)methyl)propyl)amino)-3-nitrobenzenesulfonamide);N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohex-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)-4-(((1R)-3-(morpholin-4-yl)-1-((phenylsulfanyl)methyl)propyl)amino)-3-((trifluoromethyl)sulfonyl)benzenesulfonamide;GX-070 (Obatoclax®; 1H-Indole,2-(2-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-));HGS1029; GDC-0145; GDC-0152; LCL-161; LBW-242; venetoclax; agents thattarget TRAIL or death receptors (e.g., DR4 and DR5) such as ETR2-ST01,GDC0145, HGS-1029, LBY-135, PRO-1762; drugs that target caspases,caspase-regulators, BCL-2 family members, death domain proteins, TNFfamily members, Toll family members, and/or NF-kappa-B proteins.

Angiogenesis Inhibitors:

ABT-869; AEE-788; axitinib (AG-13736); AZD-2171; CP-547,632; IM-862;pegaptamib; sorafenib; BAY43-9006; pazopanib (GW-786034); vatalanib(PTK-787, ZK-222584); sunitinib; SU-11248; VEGF trap; vandetanib;ABT-165; ZD-6474; DLL4 inhibitors.

Proteasome Inhibitors:

Bortezomib; Carfilzomib; Epoxomicin; Ixazomib; Salinosporamide A.

Kinase Inhibitors:

Afatinib; Axitinib; Bosutinib; Crizotinib; Dasatinib; Erlotinib;Fostamatinib; Gefitinib; Ibrutinib; Imatinib; Lapatinib; Lenvatinib;Mubritinib; Nilotinib; Pazopanib; Pegaptanib; Sorafenib; Sunitinib;SU6656; Vandetanib; Vemurafenib; CEP-701 (lesaurtinib); XL019;INCB018424 (ruxolitinib); ARRY-142886 (selemetinib); ARRY-438162(binimetinib); PD-325901; PD-98059; AP-23573; CCI-779; everolimus;RAD-001; rapamycin; temsirolimus; ATP-competitive TORC1/TORC2 inhibitorsincluding PI-103, PP242, PP30, Torin 1; LY294002; XL-147; CAL-120;ONC-21; AEZS-127; ETP-45658; PX-866; GDC-0941; BGT226; BEZ235; XL765.

Protein Synthesis Inhibitors:

Streptomycin; Dihydrostreptomycin; Neomycin; Framycetin; Paromomycin;Ribostamycin; Kanamycin; Amikacin; Arbekacin; Bekanamycin; Dibekacin;Tobramycin; Spectinomycin; Hygromycin B; Paromomycin; Gentamicin;Netilmicin; Sisomicin; Isepamicin; Verdamicin; Astromicin; Tetracycline;Doxycycline; Chlortetracycline; Clomocycline; Demeclocycline;Lymecycline; Meclocycline; Metacycline; Minocycline; Oxytetracycline;Penimepicycline; Rolitetracycline; Tetracycline; Glycylcyclines;Tigecycline; Oxazolidinone; Eperezolid; Linezolid; Posizolid; Radezolid;Ranbezolid; Sutezolid; Tedizolid; Peptidyl transferase inhibitors;Chloramphenicol; Azidamfenicol; Thiamphenicol; Florfenicol;Pleuromutilins; Retapamulin; Tiamulin; Valnemulin; Azithromycin;Clarithromycin; Dirithromycin; Erythromycin; Flurithromycin; Josamycin;Midecamycin; Miocamycin; Oleandomycin; Rokitamycin; Roxithromycin;Spiramycin; Troleandomycin; Tylosin; Ketolides; Telithromycin;Cethromycin; Solithromycin; Clindamycin; Lincomycin; Pirlimycin;Streptogramins; Pristinamycin; Quinupristin/dalfopristin; Virginiamycin.

Histone Deacetylase Inhibitors:

Vorinostat; Romidepsin; Chidamide; Panobinostat; Valproic acid;Belinostat; Mocetinostat; Abexinostat; Entinostat; SB939 (pracinostat);Resminostat; Givinostat; Quisinostat; thioureidobutyronitrile(Kevetrin™); CUDC-10; CHR-2845 (tefinostat); CHR-3996; 4SC-202;CG200745; ACY-1215 (rocilinostat); ME-344; sulforaphane.

Topoisomerase I Inhibitors:

camptothecin; various camptothecin derivatives and analogs (for example,NSC 100880, NSC 603071, NSC 107124, NSC 643833, NSC 629971, NSC 295500,NSC 249910, NSC 606985, NSC 74028, NSC 176323, NSC 295501, NSC 606172,NSC 606173, NSC 610458, NSC 618939, NSC 610457, NSC 610459, NSC 606499,NSC 610456, NSC 364830, and NSC 606497); morpholinisoxorubicin; SN-38.

Topoisomerase II Inihibitors:

doxorubicin; amonafide (benzisoquinolinedione); m-AMSA(4′-(9-acridinylamino)-3′-methoxymethanesulfonanilide); anthrapyrazolederivative ((NSC 355644); etoposide (VP-16); pyrazoloacridine((pyrazolo[3,4,5-kl]acridine-2(6H)-propanamine, 9-methoxy-N,N-dimethyl-5-nitro-, monomethanesulfonate); bisantrene hydrochloride;daunorubicin; deoxydoxorubicin; mitoxantrone; menogaril; N,N-dibenzyldaunomycin; oxanthrazole; rubidazone; teniposide.

DNA Intercalating Agents:

anthramycin; chicamycin A; tomaymycin; DC-81; sibiromycin;pyrrolobenzodiazepine derivative; SGD-1882((S)-2-(4-aminophenyl)-7-methoxy-8-(3S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one);SG2000 (SJG-136;(11aS,11a'S)-8,8′-(propane-1,3-diylbis(oxy))bis(7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one)).

RNA/DNA Antimetabolites:

L-alanosine; 5-azacytidine; 5-fluorouracil; acivicin; aminopterinderivative N-[2-chloro-5[[(2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino]benzoyl]L-aspartic acid(NSC 132483); aminopterin derivative N-[4-[[(2,4-diamino-5-ethyl-6-quinazolinyl)methyl]amino]benzoyl]L-aspartic acid;aminopterin derivative N-[2-chloro-4-[[(2,4-diamino-6-pteridinyl)methyl]amino]benzoyl]L-aspartic acid monohydrate;antifolate PT523((N^(α)-(4-amino-4-deoxypteroyl)-N^(γ)-hemiphthaloyl-L-ornithine));Baker's soluble antifol (NSC 139105); dichlorallyl lawsone ((2-(3,3-dichloroallyl)-3-hydroxy-1,4-naphthoquinone); brequinar; ftorafur((pro-drug; 5-fluoro-1-(tetrahydro-2-furyl)-uracil);5,6-dihydro-5-azacytidine; methotrexate; methotrexate derivative(N-[[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]-1-naphthalenyl]carbonyl]L-glutamicacid); PALA ((N-(phosphonoacetyl)-L-aspartate); pyrazofurin;trimetrexate.

DNA Antimetabolites:

3-HP; 2′-deoxy-5-fluorouridine; 5-HP; α-TGDR(α-2′-deoxy-6-thioguanosine); aphidicolin glycinate; ara C (cytosinearabinoside); 5-aza-2′-deoxycytidine; β-TGDR(β-2′-deoxy-6-thioguanosine); cyclocytidine; guanazole; hydroxyurea;inosine glycodialdehyde; macbecin II; pyrazoloimidazole; thioguanine;thiopurine.

Mitochondria Inhibitors:

pancratistatin; phenpanstatin; rhodamine-123; edelfosine;d-alpha-tocopherol succinate; compound 11β; aspirin; ellipticine;berberine; cerulenin; GX015-070 (Obatoclax®; 1H-Indole,2-(2-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-);celastrol (tripterine); metformin; Brilliant green; ME-344.

Antimitotic Agents:

allocolchicine; auristatins, such as MMAE (monomethyl auristatin E) andMMAF (monomethyl auristatin F); halichondrin B; cemadotin; colchicine;cholchicine derivative (N-benzoyl-deacetyl benzamide); dolastatin-10;dolastatin-15; maytansine; maytansinoids, such as DM1(N₂′-deacetyl-N₂′-(3-mercapto-1-oxopropyl)-maytansine); rhozoxin;paclitaxel; paclitaxel derivative((2′-N-[3-(dimethylamino)propyl]glutaramate paclitaxel); docetaxel;thiocolchicine; trityl cysteine; vinblastine sulfate; vincristinesulfate.

Nuclear Export Inhibitors:

callystatin A; delactonmycin; KPT-185 (propan-2-yl(Z)-3-[3-[3-methoxy-5-(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]prop-2-enoate);kazusamycin A; leptolstatin; leptofuranin A; leptomycin B; ratjadone;Verdinexor((Z)-3-[3-[3,5-bis(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]-N-pyridin-2-ylprop-2-enehydrazide).

hormonal therapies: anastrozole; exemestane; arzoxifene; bicalutamide;cetrorelix; degarelix; deslorelin; trilostane; dexamethasone; flutamide;raloxifene; fadrozole; toremifene; fulvestrant; letrozole; formestane;glucocorticoids; doxercalciferol; sevelamer carbonate; lasofoxifene;leuprolide acetate; megesterol; mifepristone; nilutamide; tamoxifencitrate; abarelix; prednisone; finasteride; rilostane; buserelin;luteinizing hormone releasing hormone (LHRH); Histrelin; trilostane ormodrastane; fosrelin; goserelin.

Any of these agents that include, or that may be modified to include, asite of attachment to an antibody and/or binding fragment may beincluded in an anti-huLRRC15 ADC.

Data presented herein demonstrate that anti-huLRRC15 ADCs exert potentanti-tumor activity against huLRRC15 stromal(+)/cancer(−) tumors that ismediated, at least in part, by a targeted bystander killing effect. Forexample, as demonstrated in FIG. 14A, cells expressing huLRRC15 aresensitive in vitro to anti-huLRRC15 ADCs containing either monomethylauristatin E (“MMAE”) or monomethyl auristatin F (“MMAF”) as the drug.However, in vivo, huLRRC15 stromal(+)/cancer (−) PANC-1 xenografts aresensitive to the MMAE anti-huLRRC15 ADC, whereas the MMAF anti-huLRRC15ADC does not shrink tumors, as demonstrated in FIG. 17B. This is alsoobserved with huLRRC15 stromal(+)/cancer (−) EBC-1 xenografts, asdemonstrated in FIG. 15A. MMAE released from the ADCs internalized inhuLRRC15-expressing stromal cells is cell permeable, allowing targetedbystander killing of cancer cells adjacent to the stromal cells. MMAF isnot cell permeable, is retained within the huLRRC15-expressing stromalcells, and does not migrate to kill neighboring cancer cells. ThehuLRRC15-expressing stromal cells divide at a much slower rate thancancer cells in vivo and are therefore intrinsically less sensitive thanrapidly dividing cancer cells to anti-mitotic agents such as MMAE andMMAF (as shown in FIG. 16). Together, these data demonstrate thatanti-huLRRC15 ADCs containing cell-permeating cytostatic and/orcytotoxic agents exert anti-tumor activity against huLRRC15stromal(+)/cancer (−) tumors via targeted bystander killing and areuseful therapeutically for the treatment of huLRRC15 stromal(+)/cancer(−) tumors.

Accordingly, in some embodiments, the cytotoxic and/or cytostatic agentsincluded in an anti-huLRRC15 ADC will, upon cleavage of the ADC, be ableto traverse cell membranes (“cell permeable cytostatic and/or cytotoxicagents”). Specific cytotoxic and/or cytostatic agents of interest,and/or cleavage products of ADCs including such agents, may be testedfor the ability to traverse cell membranes using routine methods knownto those of skill in the art. Permeability (P) of molecules across amembrane can be expressed as P=KD/Δx where K is the partitioncoefficient, D is the diffusion coefficient, and Δx is the thickness ofthe cell membrane. The diffusion coefficient (D) is a measure of therate of entry into the cytoplasm depending on the molecular weight orsize of a molecule. K is a measure of the solubility of the substance inlipids. A low value of K describes a molecule like water that is notsoluble in lipid. Graphically, it is expected that permeability (P) as afunction of the partition coefficient (K) will increase linearly when Dand Δx are constants. (Walter & Gutknecht, 1986, “Permeability of smallnonelectrolytes through lipid bilayer membranes,” Journal of MembraneBiology 90:207-217; Diamond & Katz, 1974, “Interpretation ofnonelectrolyte partition coefficients between dimyristoyl lecithin andwater,” Journal of Membrane Biology 17:121-154).

In a specific embodiment, the cytotoxic and/or cytostatic agent is acell-permeable antimitotic agent.

In another specific embodiment, the cytotoxic and/or cytostatic agent isa cell-permeable auristatin, such as, for example, dolastatin-10 orMMAE.

In another specific embodiment, the cytotoxic and/or cytostatic agent isa cell-permeable minor groove-binding DNA cross-linking agent, such as,for example, a pyrrolobenzodiazepine (“PBD”) dimer.

7.4.2. Linkers

In the anti-huLRRC15 ADCs described herein, the cytotoxic and/orcytostatic agents are linked to the antigen binding moiety by way oflinkers. The linkers may be short, long, hydrophobic, hydrophilic,flexible or rigid, or may be composed of segments that eachindependently have one or more of the above-mentioned properties suchthat the linker may include segments having different properties. Thelinkers may be polyvalent such that they covalently link more than oneagent to a single site on the antibody, or monovalent such thatcovalently they link a single agent to a single site on the antibody.

As will be appreciated by skilled artisans, the linkers link thecytotoxic and/or cytostatic agents to the antigen binding moiety byforming a covalent linkage to the cytotoxic and/or cytostatic agent atone location and a covalent linkage to the antigen binding moiety atanother. The covalent linkages are formed by reaction between functionalgroups on the linker and functional groups on the agents and the antigenbinding moiety. As used herein, the expression “linker” is intended toinclude (i) unconjugated forms of the linker that include a functionalgroup capable of covalently linking the linker to a cytotoxic and/orcytostatic agent and a functional group capable of covalently linkingthe linker to the antigen binding moiety such as an antibody; (ii)partially conjugated forms of the linker that includes a functionalgroup capable of covalently linking the linker to an antigen bindingmoiety such as an antibody and that is covalently linked to a cytotoxicand/or cytostatic agent, or vice versa; and (iii) fully conjugated formsof the linker that is covalently linked to both a cytotoxic and/orcytostatic agent and an antigen binding moiety such as an antibody. Insome specific embodiments of linkers and ADCs described herein, as wellas synthons used to conjugate linker-agents to antibodies, moietiescomprising the functional groups on the linker and covalent linkagesformed between the linker and antibody are specifically illustrated asR^(x) and XY, respectively.

The linkers are preferably, but need not be, chemically stable toconditions outside the cell, and may be designed to cleave, immolateand/or otherwise specifically degrade inside the cell. Alternatively,linkers that are not designed to specifically cleave or degrade insidethe cell may be used. Particular linkers may also be processedextracellularly in the tumor microenvironment by enzymes present at highlevels in tumor stroma. Choice of stable versus unstable linker maydepend upon the toxicity of the cytotoxic and/or cytostatic agent. Awide variety of linkers useful for linking drugs to antibodies in thecontext of ADCs are known in the art. Any of these linkers, as well asother linkers, may be used to link the cytotoxic and/or cytostaticagents to the antibody of the ADCs described herein.

Exemplary polyvalent linkers that may be used to link many cytotoxicand/or cytostatic agents to a single antibody molecule are described,for example, in WO 2009/073445; WO 2010/068795; WO 2010/138719; WO2011/120053; WO 2011/171020; WO 2013/096901; WO 2014/008375; WO2014/093379; WO 2014/093394; WO 2014/093640, the contents of which areincorporated herein by reference in their entireties. For example, theFleximer linker technology developed by Mersana et al. has the potentialto enable high-DAR ADCs with good physicochemical properties. As shownbelow, the Mersana technology is based on incorporating drug moleculesinto a solubilizing poly-acetal backbone via a sequence of ester bonds.The methodology renders highly-loaded ADCs (DAR up to 20) whilemaintaining good physicochemical properties.

Additional examples of dendritic type linkers can be found in US2006/116422; US 2005/271615; de Groot et al (2003) Angew. Chem. Int. Ed.42:4490-4494; Amir et al (2003) Angew. Chem. Int. Ed. 42:4494-4499;Shamis et al (2004) J. Am. Chem. Soc. 126:1726-1731; Sun et al (2002)Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003)Bioorganic & Medicinal Chemistry 11:1761-1768; King et al (2002)Tetrahedron Letters 43:1987-1990, each of which is incorporated hereinby reference.

Exemplary monovalent linkers that may be used are described, forexample, in Nolting, 2013, Antibody-Drug Conjugates, Methods inMolecular Biology 1045:71-100; Kitson et al., 2013, CROs/CMOs—ChemicaOggi—Chemistry Today 31(4):30-38; Ducry et al., 2010, Bioconjugate Chem.21:5-13; Zhao et al., 2011, J. Med. Chem. 54:3606-3623; U.S. Pat. No.7,223,837; U.S. Pat. No. 8,568,728; U.S. Pat. No. 8,535,678; andWO2004010957, each of which is incorporated herein by reference.

By way of example and not limitation, some cleavable and noncleavablelinkers that may be included in the anti-huLRRC15 ADCs described hereinare described below.

7.4.2.1. Cleavable Linkers

In certain embodiments, the linker selected is cleavable in vivo.Cleavable linkers may include chemically or enzymatically unstable ordegradable linkages. Cleavable linkers generally rely on processesinside the cell to liberate the drug, such as reduction in thecytoplasm, exposure to acidic conditions in the lysosome, or cleavage byspecific proteases or other enzymes within the cell. Cleavable linkersgenerally incorporate one or more chemical bonds that are eitherchemically or enzymatically cleavable while the remainder of the linkeris noncleavable. In certain embodiments, a linker comprises a chemicallylabile group such as hydrazone and/or disulfide groups. Linkerscomprising chemically labile groups exploit differential propertiesbetween the plasma and some cytoplasmic compartments. The intracellularconditions to facilitate drug release for hydrazone containing linkersare the acidic environment of endosomes and lysosomes, while thedisulfide containing linkers are reduced in the cytosol, which containshigh thiol concentrations, e.g., glutathione. In certain embodiments,the plasma stability of a linker comprising a chemically labile groupmay be increased by introducing steric hindrance using substituents nearthe chemically labile group.

Acid-labile groups, such as hydrazone, remain intact during systemiccirculation in the blood's neutral pH environment (pH 7.3-7.5) andundergo hydrolysis and release the drug once the ADC is internalizedinto mildly acidic endosomal (pH 5.0-6.5) and lysosomal (pH 4.5-5.0)compartments of the cell. This pH dependent release mechanism has beenassociated with nonspecific release of the drug. To increase thestability of the hydrazone group of the linker, the linker may be variedby chemical modification, e.g., substitution, allowing tuning to achievemore efficient release in the lysosome with a minimized loss incirculation.

Hydrazone-containing linkers may contain additional cleavage sites, suchas additional acid-labile cleavage sites and/or enzymatically labilecleavage sites. ADCs including exemplary hydrazone-containing linkersinclude the following structures:

wherein D and Ab represent the cytotoxic and/or cytostatic agent (drug)and antibody, respectively, and n represents the number of drug-linkerslinked to the antibody. In certain linkers such as linker (Ig), thelinker comprises two cleavable groups—a disulfide and a hydrazonemoiety. For such linkers, effective release of the unmodified free drugrequires acidic pH or disulfide reduction and acidic pH. Linkers such as(Ih) and (Ii) have been shown to be effective with a single hydrazonecleavage site.

Other acid-labile groups that may be included in linkers includecis-aconityl-containing linkers. cis-Aconityl chemistry uses acarboxylic acid juxtaposed to an amide bond to accelerate amidehydrolysis under acidic conditions.

Cleavable linkers may also include a disulfide group. Disulfides arethermodynamically stable at physiological pH and are designed to releasethe drug upon internalization inside cells, wherein the cytosol providesa significantly more reducing environment compared to the extracellularenvironment. Scission of disulfide bonds generally requires the presenceof a cytoplasmic thiol cofactor, such as (reduced) glutathione (GSH),such that disulfide-containing linkers are reasonably stable incirculation, selectively releasing the drug in the cytosol. Theintracellular enzyme protein disulfide isomerase, or similar enzymescapable of cleaving disulfide bonds, may also contribute to thepreferential cleavage of disulfide bonds inside cells. GSH is reportedto be present in cells in the concentration range of 0.5-10 mM comparedwith a significantly lower concentration of GSH or cysteine, the mostabundant low-molecular weight thiol, in circulation at approximately 5μM. Tumor cells, where irregular blood flow leads to a hypoxic state,result in enhanced activity of reductive enzymes and therefore evenhigher glutathione concentrations. In certain embodiments, the in vivostability of a disulfide-containing linker may be enhanced by chemicalmodification of the linker, e.g., use of steric hinderance adjacent tothe disulfide bond.

ADCs including exemplary disulfide-containing linkers include thefollowing structures:

wherein D and Ab represent the drug and antibody, respectively, nrepresents the number of drug-linkers linked to the antibody, and R isindependently selected at each occurrence from hydrogen or alkyl, forexample. In certain embodiments, increasing steric hinderance adjacentto the disulfide bond increases the stability of the linker. Structuressuch as (Ij) and (Il) show increased in vivo stability when one or moreR groups is selected from a lower alkyl such as methyl.

Another type of cleavable linker that may be used is a linker that isspecifically cleaved by an enzyme. Such linkers are typicallypeptide-based or include peptidic regions that act as substrates forenzymes. Peptide based linkers tend to be more stable in plasma andextracellular milieu than chemically labile linkers. Peptide bondsgenerally have good serum stability, as lysosomal proteolytic enzymeshave very low activity in blood due to endogenous inhibitors and theunfavorably high pH value of blood compared to lysosomes. Release of adrug from an antibody occurs specifically due to the action of lysosomalproteases, e.g., cathepsin and plasmin. These proteases may be presentat elevated levels in certain tumor cells.

In exemplary embodiments, the cleavable peptide is selected fromtetrapeptides such as Gly-Phe-Leu-Gly (SEQ ID NO:80), Ala-Leu-Ala-Leu(SEQ ID NO:81) or dipeptides such as Val-Cit, Val-Ala, Met-(D)Lys,Asn-(D)Lys, Val-(D)Asp, Phe-Lys, Ile-Val, Asp-Val, His-Val,NorVal-(D)Asp, Ala-(D)Asp, Met-Lys, Asn-Lys, Ile-Pro, Me3Lys-Pro,PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys,Asn-(D)Lys, Met-(D)Lys, Asn-(D)Lys. In certain embodiments, dipeptidesare preferred over longer polypeptides due to hydrophobicity of thelonger peptides.

A variety of dipeptide-based cleavable linkers useful for linking drugssuch as doxorubicin, mitomycin, campotothecin, tallysomycin andauristatin/auristatin family members to antibodies have been described(see, Dubowchik et al., 1998, J Org. Chem. 67:1866-1872; Dubowchik etal., 1998, Bioorg. Med. Chem. Lett. 8(21):3341-3346; Walker et al.,2002, Bioorg. Med. Chem. Lett. 12:217-219; Walker et al., 2004, Bioorg.Med. Chem. Lett. 14:4323-4327; and Francisco et al., 2003, Blood102:1458-1465, Dornina et al., 2008, Bioconjugate Chemistry19:1960-1963, of each of which is incorporated herein by reference). Allof these dipeptide linkers, or modified versions of these dipeptidelinkers, may be used in the ADCs described herein. Other dipeptidelinkers that may be used include those found in ADCs such as SeattleGenetics' Brentuximab Vendotin SGN-35 (Adcetris™), Seattle GeneticsSGN-75 (anti-CD-70, Val-Cit-MMAF), Celldex Therapeutics glembatumumab(CDX-011) (anti-GPNMB, Val-Cit-MMAE), and Cytogen PSMA-ADC(PSMA-ADC-1301) (anti-PSMA, Val-Cit-MMAE).

Enzymatically cleavable linkers may include a self-immolative spacer tospatially separate the drug from the site of enzymatic cleavage. Thedirect attachment of a drug to a peptide linker can result inproteolytic release of an amino acid adduct of the drug, therebyimpairing its activity. The use of a self-immolative spacer allows forthe elimination of the fully active, chemically unmodified drug uponamide bond hydrolysis.

One self-immolative spacer is the bifunctional para-aminobenzyl alcoholgroup, which is linked to the peptide through the amino group, formingan amide bond, while amine containing drugs may be attached throughcarbamate functionalities to the benzylic hydroxyl group of the linker(PABC). The resulting prodrugs are activated upon protease-mediatedcleavage, leading to a 1,6-elimination reaction releasing the unmodifieddrug, carbon dioxide, and remnants of the linker group. The followingscheme depicts the fragmentation of p-amidobenzyl ether and release ofthe drug:

wherein X-D represents the unmodified drug.

Heterocyclic variants of this self-immolative group have also beendescribed. See for example, U.S. Pat. No. 7,989,434, incorporated hereinby reference.

In some embodiments, the enzymatically cleavable linker is aß-glucuronic acid-based linker. Facile release of the drug may berealized through cleavage of the ß-glucuronide glycosidic bond by thelysosomal enzyme ß-glucuronidase. This enzyme is present abundantlywithin lysosomes and is overexpressed in some tumor types, while theenzyme activity outside cells is low. ß-Glucuronic acid-based linkersmay be used to circumvent the tendency of an ADC to undergo aggregationdue to the hydrophilic nature of ß-glucuronides. In some embodiments,ß-glucuronic acid-based linkers are preferred as linkers for ADCs linkedto hydrophobic drugs. The following scheme depicts the release of thedrug from and ADC containing a ß-glucuronic acid-based linker:

A variety of cleavable ß-glucuronic acid-based linkers useful forlinking drugs such as auristatins, camptothecin and doxorubicinanalogues, CBI minor-groove binders, and psymberin to antibodies havebeen described (see, see Nolting, Chapter 5 “Linker Technology inAntibody-Drug Conjugates,” In: Antibody-Drug Conjugates: Methods inMolecular Biology, vol. 1045, pp. 71-100, Laurent Ducry (Ed.), SpringerScience & Business Medica, LLC, 2013; Jeffrey et al., 2006, Bioconjug.Chem. 17:831-840; Jeffrey et al., 2007, Bioorg. Med. Chem. Lett.17:2278-2280; and Jiang et al., 2005, J Am. Chem. Soc. 127:11254-11255,each of which is incorporated herein by reference). All of theseß-glucuronic acid-based linkers may be used in the anti-huLRRC15 ADCsdescribed herein.

Additionally, cytotoxic and/or cytostatic agents containing a phenolgroup can be covalently bonded to a linker through the phenolic oxygen.One such linker, described in WO 2007/089149, relies on a methodogy inwhich a diamino-ethane “SpaceLink” is used in conjunction withtraditional “PABO”-based self-immolative groups to deliver phenols. Thecleavage of the linker is depicted schematically below, where Drepresents a cytotoxic and/or cytostatic agent having a phenolichydroxyl group.

Cleavable linkers may include noncleavable portions or segments, and/orcleavable segments or portions may be included in an otherwisenon-cleavable linker to render it cleavable. By way of example only,polyethylene glycol (PEG) and related polymers may include cleavablegroups in the polymer backbone. For example, a polyethylene glycol orpolymer linker may include one or more cleavable groups such as adisulfide, a hydrazone or a dipeptide.

Other degradable linkages that may be employed in linkers include, butare not limited to, ester linkages formed by the reaction of PEGcarboxylic acids or activated PEG carboxylic acids with alcohol groupson a biologically active agent, wherein such ester groups generallyhydrolyze under physiological conditions to release the biologicallyactive agent. Hydrolytically degradable linkages include, but are notlimited to, carbonate linkages; imine linkages resulting from reactionof an amine and an aldehyde; phosphate ester linkages formed by reactingan alcohol with a phosphate group; acetal linkages that are the reactionproduct of an aldehyde and an alcohol; orthoester linkages that are thereaction product of a formate and an alcohol; and oligonucleotidelinkages formed by a phosphoramidite group, including but not limitedto, at the end of a polymer, and a 5′-hydroxyl group of anoligonucleotide.

In certain embodiments, the linker comprises an enzymatically cleavablepeptide moiety, for example, a linker comprising structural formula(IVa), (IVb), (IVc), or (IVd):

or a salt thereof, wherein:

-   -   peptide represents a peptide (illustrated C→N and not showing        the carboxy and amino “termini”) cleavable by a lysosomal        enzyme;    -   T represents a polymer comprising one or more ethylene glycol        units or an alkylene chain, or combinations thereof;    -   R^(a) is selected from hydrogen, alkyl, sulfonate and methyl        sulfonate;    -   p is an integer ranging from 0 to 5;    -   q is 0 or 1;    -   x is 0 or 1;    -   y is 0 or 1;    -   represents the point of attachment of the linker to a cytotoxic        and/or cytostatic agent; and    -   * represents the point of attachment to the remainder of the        linker.

In certain embodiments, the peptide is selected from a tripeptide or adipeptide. In particular embodiments, the dipeptide is selected from:Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit;Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp;Ala-Val; Val-Ala; Phe-Lys; Val-Lys; Ala-Lys; Phe-Cit; Leu-Cit; Ile-Cit;Phe-Arg; and Trp-Cit.

Specific exemplary embodiments of linkers according to structuralformula (IVa) that may be included in the ADCs described herein includethe linkers illustrated below (as illustrated, the linkers include agroup suitable for covalently linking the linker to an antibody):

Specific exemplary embodiments of linkers according to structuralformula (IVb) that may be included in the ADCs described herein includethe linkers illustrated below (as illustrated, the linkers include agroup suitable for covalently linking the linker to an antibody):

Specific exemplary embodiments of linkers according to structuralformula (IVc) that may be included in the ADCs described herein includethe linkers illustrated below (as illustrated, the linkers include agroup suitable for covalently linking the linker to an antibody):

Specific exemplary embodiments of linkers according to structuralformula (IVd) that may be included in the ADCs described herein includethe linkers illustrated below (as illustrated, the linkers include agroup suitable for covalently linking the linker to an antibody):

In certain embodiments, the linker comprising structural formula (IVa),(IVb), (IVc), or (IVd) further comprises a carbonate moiety cleavable byexposure to an acidic medium. In particular embodiments, the linker isattached through an oxygen to a cytotoxic and/or cytostatic agent.

7.4.2.2. Non-Cleavable Linkers

Although cleavable linkers may provide certain advantages, the linkerscomposing the ADC described herein need not be cleavable. Fornon-cleavable linkers, the release of drug does not depend on thedifferential properties between the plasma and some cytoplasmiccompartments. The release of the drug is postulated to occur afterinternalization of the ADC via antigen-mediated endocytosis and deliveryto lysosomal compartment, where the antibody is degraded to the level ofamino acids through intracellular proteolytic degradation. This processreleases a drug derivative, which is formed by the drug, the linker, andthe amino acid residue to which the linker was covalently attached. Theamino acid drug metabolites from conjugates with non-cleavable linkersare more hydrophilic and generally less membrane permeable, which leadsto less bystander effects and less nonspecific toxicities compared toconjugates with a cleavable linker. In general, ADCs with noncleavablelinkers have greater stability in circulation than ADCs with cleavablelinkers. Non-cleavable linkers may be alkylene chains, or may bepolymeric in nature, such as, for example, those based upon polyalkyleneglycol polymers, amide polymers, or may include segments of alkylenechains, polyalkylene glycols and/or amide polymers.

A variety of non-cleavable linkers used to link drugs to antibodies havebeen described. See, Jeffrey et al., 2006, Bioconjug. Chem. 17; 831-840;Jeffrey et al., 2007, Bioorg. Med. Chem. Lett. 17:2278-2280; and Jianget al., 2005, J. Am. Chem. Soc. 127:11254-11255, each of which isincorporated herein by reference. All of these linkers may be includedin the ADCs described herein.

In certain embodiments, the linker is non-cleavable in vivo, for examplea linker according to structural formula (VIa), (VIb), (VIc) or (VId)(as illustrated, the linkers include a group suitable for covalentlylinking the linker to an antibody:

or salts thereof, wherein:

-   -   R^(a) is selected from hydrogen, alkyl, sulfonate and methyl        sulfonate;    -   R^(x) is a moiety including a functional group capable of        covalently linking the linker to an antibody; and

represents the point of attachment of the linker to a cytotoxic and/orcytostatic agent.

Specific exemplary embodiments of linkers according to structuralformula (VIa)-(VId) that may be included in the ADCs described hereininclude the linkers illustrated below (as illustrated, the linkersinclude a group suitable for covalently linking the linker to anantibody, and “

” represents the point of attachment to a cytotoxic and/or cytostaticagent):

7.4.2.3. Groups Used to Attach Linkers to Antibodies

A variety of groups may be used to attach linker-drug synthons toantibodies to yield ADCs. Attachment groups can be electrophilic innature and include: maleimide groups, activiated disulfides, activeesters such as NHS esters and HOBt esters, haloformates, acid halides,alkyl and benzyl halides such as haloacetamides. As discussed below,there are also emerging technologies related to “self-stabilizing”maleimides and “bridging disulfides” that can be used in accordance withthe disclosure. The specific group used will depend, in part, on thesite of attachment to the antibody.

One example of a “self-stabilizing” maleimide group that hydrolyzesspontaneously under antibody conjugation conditions to give an ADCspecies with improved stability is depicted in the schematic below. SeeUS20130309256 A1; also Lyon et al., Nature Biotech published online,doi:10.1038/nbt.2968).

Polytherics has disclosed a method for bridging a pair of sulfhydrylgroups derived from reduction of a native hinge disulfide bond. See,Badescu et al., 2014, Bioconjugate Chem. 25:1124-1136. The reaction isdepicted in the schematic below. An advantage of this methodology is theability to synthesize homogeneous DAR4 ADCs by full reduction of IgGs(to give 4 pairs of sulfhydryls) followed by reaction with 4 equivalentsof the alkylating agent. ADCs containing “bridged disulfides” are alsoclaimed to have increased stability.

Similarly, as depicted below, a maleimide derivative (1, below) that iscapable of bridging a pair of sulfhydryl groups has been developed. SeeWO2013/085925.

7.4.2.4. Linker Selection Considerations

As is known by skilled artisans, the linker selected for a particularADC may be influenced by a variety of factors, including but not limitedto, the site of attachment to the antibody (e.g., Lys, Cys or otheramino acid residues), structural constraints of the drug pharmacophoreand the lipophilicity of the drug. The specific linker selected for anADC should seek to balance these different factors for the specificantibody/drug combination. For a review of the factors that areinfluenced by choice of linkers in ADCs, see Nolting, Chapter 5 “LinkerTechnology in Antibody-Drug Conjugates,” In: Antibody-Drug Conjugates:Methods in Molecular Biology, vol. 1045, pp. 71-100, Laurent Ducry(Ed.), Springer Science & Business Medica, LLC, 2013.

For example, as discussed above, anti-huLRRC15 ADCs have been observedto induce bystander killing of cancer cells present in the vicinity ofhuLRRC15-expressing stromal cells for huLRRC15 stromal(+)/cancer (−)tumors. The mechanism of bystander cell killing by ADCs has indicatedthat metabolic products formed during intracellular processing of theADCs may play a role. Cell-permeable cytotoxic and/or cytostaticmetabolites generated by metabolism of the ADCs in huLRRC15-expressingcells appear to play a role in bystander cell killing, whilenon-cell-permeable metabolites, which are incapable of traversing thecell membrane and diffusing into the medium cannot effect bystanderkilling. In certain embodiments, the linker is selected to effect,enhance or increase the bystander killing effect of the anti-huLRRC15ADCs.

The properties of the linker may also impact aggregation of the ADCunder conditions of use and/or storage. Typically, ADCs reported in theliterature contain no more than 3-4 drug molecules per antigen-bindingmoiety, for example, per antibody molecule (see, e.g., Chari, 2008, AccChem Res 41:98-107). Attempts to obtain higher drug-to-antibody ratios(“DAR”) often failed, particularly if both the drug and the linker werehydrophobic, due to aggregation of the ADC (King et al., 2002, J MedChem 45:4336-4343; Hollander et al., 2008, Bioconjugate Chem 19:358-361;Burke et al., 2009 Bioconjugate Chem 20:1242-1250). In many instances,DARs higher than 3-4 could be beneficial as a means of increasingpotency. In instances where the cytotoxic and/or cytostatic agent ishydrophobic in nature, it may be desirable to select linkers that arerelatively hydrophilic as a means of reducing ADC aggregation,especially in instances where DARS greater than 3-4 are desired. Thus,in certain embodiments, the linker incorporates chemical moieties thatreduce aggregation of the ADCs during storage and/or use. A linker mayincorporate polar or hydrophilic groups such as charged groups or groupsthat become charged under physiological pH to reduce the aggregation ofthe ADCs. For example, a linker may incorporate charged groups such assalts or groups that deprotonate, e.g., carboxylates, or protonate,e.g., amines, at physiological pH.

Exemplary polyvalent linkers that have been reported to yield DARs ashigh as 20 that may be used to link numerous cytotoxic and/or cytostaticagents to an antibody are described in WO 2009/073445; WO 2010/068795;WO 2010/138719; WO 2011/120053; WO 2011/171020; WO 2013/096901; WO2014/008375; WO 2014/093379; WO 2014/093394; WO 2014/093640, thecontents of which are incorporated herein by reference in theirentireties.

In particular embodiments, the aggregation of the ADCs during storage oruse is less than about 10% as determined by size-exclusionchromatography (SEC). In particular embodiments, the aggregation of theADCs during storage or use is less than 10%, such as less than about 5%,less than about 4%, less than about 3%, less than about 2%, less thanabout 1%, less than about 0.5%, less than about 0.1%, or even lower, asdetermined by size-exclusion chromatography (SEC). In particularembodiments, the aggregation of the ADCs during storage or use is in arange of any two of the foregoing values, such as but not limited tofrom about 0.1% to 10%, 0.1% to 5%, 0.5% to 10%, 0.5% to 5%, or 1% to10%.

7.4.3. Embodiments of Anti-huLRRC15 Antibody Drug Conjugates

As described above, embodiments of an anti-huLRRC15 ADC includecompounds having a structure according to formula (I): [D-L-XY-]_(n)-Ab(I), or a salt thereof, wherein D is the cytotoxic and/or cytostaticagent; L is the linker; Ab is the antibody; XY represents a covalentlinkage linking linker L to antibody Ab; and n is an integer rangingfrom 2 to 8.

In some embodiments, XY is a linkage formed with an amino group onantibody Ab, such as an amide or a thiourea, or a linkage formed with asulfydryl group on antibody Ab, such as a thioether. In certain suchembodiments, XY is a thioether.

In some embodiments, L comprises Val-Cit or Val-Ala.

In some embodiments, the compound according to structural formula (I)has a structure of formula (IIa):

In some embodiments, the compound according to structural formula (I)has a structure of formula (IIb):

In some embodiments of the compound of formula (I), (IIa), or (IIb), Dis an antimitotic agent or a DNA-intercalating agent. In some suchembodiments, D is an antimitotic agent which is a cell-permeableantimitotic agent. In certain such embodiments, the cell-permeableantimitotic agent is MMAE. In other such embodiments, D is aDNA-intercalating agent which is a pyrrolobenzodiazepine (PBD) dimer.

In some embodiments, the compound according to structural formula (I)has a structure of formula (IIIa):

In some embodiments, the compound according to structural formula (I)has a structure of formula (IIIb):

In some embodiments of the compound of formula (I), (IIa), (IIb),(IIIa), or (IIIb), Ab is an antibody comprising three V_(H) CDRscorresponding in sequence, respectively, to SEQ ID NO:10, SEQ ID NO:11and SEQ ID NO:12 and three V_(L) CDRs corresponding in sequence,respectively, to SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15. In somesuch embodiments, Ab is an antibody with a V_(H) having an amino acidsequence of SEQ ID NO:16, and a V_(L) having an amino acid sequence ofSEQ ID NO:17. In other embodiments, Ab is an antibody comprising threeV_(H) CDRs corresponding in sequence, respectively, to SEQ ID NO:20, SEQID NO:21 and SEQ ID NO:22 and three V_(L) CDRs corresponding insequence, respectively, to SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25.In some such embodiments, Ab is an antibody with a V_(H) having an aminoacid sequence of SEQ ID NO:26, and a V_(L) having an amino acid sequenceof SEQ ID NO:27. In some embodiments, Ab is a human IgG₁. In some suchembodiments, Ab is an antibody with a heavy chain having an amino acidsequence of SEQ ID NO:18 or 102, and a light chain having an amino acidsequence of SEQ ID NO:19. In other such embodiments, Ab is an antibodywith a heavy chain having an amino acid sequence of SEQ ID NO:100 or103, and a light chain having an amino acid sequence of SEQ ID NO:19. Inother such embodiments, Ab is an antibody with a heavy chain having anamino acid sequence of SEQ ID NO:28 or 101, and a light chain having anamino acid sequence of SEQ ID NO:29. In other such embodiments, Ab is anantibody with a heavy chain having an amino acid sequence of SEQ IDNO:104 or 105, and a light chain having an amino acid sequence of SEQ IDNO:29. In some embodiments, Ab is an antibody selected from huM25,huM25-S239C, huAD208.4.1, and huAD208.4.1-S239C. In certain suchembodiments, Ab is huM25. In other such embodiments, Ab is huM25-S239C.In yet other such embodiments, Ab is huAD208.4.1. In yet other suchembodiments, Ab is huAD208.4.1-S239C.

In some embodiments of the compound of formula (I), (IIa), (IIb),(IIIa), or (IIIb), n is 2, 3, or 4. In certain such embodiments, n is 2or 4.

7.5. Methods of Making Anti-huLRRC15 Antibody Drug Conjugates

The ADCs described herein may be synthesized using chemistries that arewell-known. The chemistries selected will depend upon, among otherthings, the identity of the cytotoxic and/or cytostatic agent(s), thelinker and the groups used to attach linker to the antibody. Generally,ADCs according to formula (I) may be prepared according to the followingscheme:D-L-R^(x)+Ab-R^(y)→(I)[D-L-XY]_(n)-Ab

where D, L, Ab, XY and n are as previously defined, and R^(x) and R^(y)represent complementary groups capable of forming covalent linkages withone another, as discussed above.

The identities of groups R^(x) and R^(y) will depend upon the chemistryused to link synthon D-L-R^(x) to the antibody. Generally, the chemistryused should not alter the integrity of the antibody, for example itsability to bind its target. Preferably, the binding properties of theconjugated antibody will closely resemble those of the unconjugatedantibody. A variety of chemistries and techniques for conjugatingmolecules to biological molecules such as antibodies are known in theart and in particular to antibodies, are well-known. See, e.g., Amon etal., “Monoclonal Antibodies For Immunotargeting Of Drugs In CancerTherapy,” in: Monoclonal Antibodies And Cancer Therapy, Reisfeld et al.Eds., Alan R. Liss, Inc., 1985; Hellstrom et al., “Antibodies For DrugDelivery,” in: Controlled Drug Delivery, Robinson et al. Eds., MarcelDekker, Inc., 2nd Ed. 1987; Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review,” in: Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al., Eds., 1985;“Analysis, Results, and Future Prospective of the Therapeutic Use ofRadiolabeled Antibody In Cancer Therapy,” in: Monoclonal Antibodies ForCancer Detection And Therapy, Baldwin et al., Eds., Academic Press,1985; Thorpe et al., 1982, Immunol. Rev. 62:119-58; PCT publication WO89/12624. Any of these chemistries may be used to link the synthons toan antibody.

A number of functional groups R^(x) and chemistries useful for linkingsynthons to accessible lysine residues are known, and include by way ofexample and not limitation NHS-esters and isothiocyanates.

A number of functional groups R^(x) and chemistries useful for linkingsynthons to accessible free sulfhydryl groups of cysteine residues areknown, and include by way of example and not limitation haloacetyls andmaleimides.

However, conjugation chemistries are not limited to available side chaingroups. Side chains such as amines may be converted to other usefulgroups, such as hydroxyls, by linking an appropriate small molecule tothe amine. This strategy can be used to increase the number of availablelinking sites on the antibody by conjugating multifunctional smallmolecules to side chains of accessible amino acid residues of theantibody. Functional groups R^(x) suitable for covalently linking thesynthons to these “converted” functional groups are then included in thesynthons.

An antibody may also be engineered to include amino acid residues forconjugation. An approach for engineering antibodies to includenon-genetically encoded amino acid residues useful for conjugating drugsin the context of ADCs is described by Axup et al., 2012, Proc Natl AcadSci USA. 109(40):16101-16106, as are chemistries and functional groupsuseful for linking synthons to the non-encoded amino acids.

Typically, the synthons are linked to the side chains of amino acidresidues of the antibody, including, for example, the primary aminogroup of accessible lysine residues or the sulfhydryl group ofaccessible cysteine residues. Free sulfhydryl groups may be obtained byreducing interchain disulfide bonds.

For linkages where R^(y) is a sulfhydryl group (for example, when R^(x)is a maleimide), the antibody is generally first fully or partiallyreduced to disrupt interchain disulfide bridges between cysteineresidues. Specific cysteine residues and interchain disulfide bridgesthat may be reduced for attachment of drug-linker synthons including agroup suitable for conjugation to a sulfhydryl group for exemplaryantibodies huM25, huAD208.4.1, huAD208.12.1, huAD208.14.1, hu139.10,muAD208.9.1, muAD210.40.9, include by way of example and not limitation,residues C233, C239, and C242 (Kabat numbering system; corresponding toresidues C220, C226, and C229 Eu numbering) on the human IgG₁ heavychain, and residue C214 (Kabat numbering system) on the human Ig kappalight chain.

Cysteine residues for synthon attachment that do not participate indisulfide bridges may be engineered into an antibody by mutation of oneor more codons. Reducing these unpaired cysteines yields a sulfhydrylgroup suitable for conjugation. Preferred positions for incorporatingengineered cysteines include, by way of example and not limitation,positions S112C, S113C, A114C, S115C, A176C, S180C, S239C, S252C, V286C,V292C, S357C, A359C, S398C, S428C (Kabat numbering) on the human IgG₁heavy chain and positions V110C, S114C, S121C, S127C, S168C, V205C(Kabat numbering) on the human Ig kappa light chain (see, e.g., U.S.Pat. No. 7,521,541, U.S. Pat. No. 7,855,275 and U.S. Pat. No.8,455,622).

Mutation of a cysteine residue known to participate in an existingdisulfide bridge may also be engineered such that the resulting unpairedcysteine partner is available to form a sulfide linker to a drug.Examples of engineered cysteine mutations include, but are not limitedto, light chain constant C214 mutations, for example C214A.

As will be appreciated by skilled artisans, the number of cytotoxicand/or cytostatic agents linked to an antibody molecule may vary, suchthat an ADC preparation may be heterogeneous in nature, where someantibodies in the preparation contain one linked agent, some two, somethree, etc. (and some none). The degree of heterogeneity will dependupon, among other things, the chemistries used for linking the cytotoxicand/or cytostatic agents. For example, where the antibodies are reducedto yield sulfhydryl groups for attachment, heterogenous mixtures ofantibodies having zero, 2, 4, 6 or 8 linked agents per molecule areoften produced. Furthermore, by limiting the molar ratio of attachmentcompound, antibodies having zero, 1, 2, 3, 4, 5, 6, 7 or 8 linked agentsper molecule are often produced. Thus, it will be understood thatdepending upon context, stated drug antibody ratios (DARs) may beaverages for a collection of antibodies. For example, “DAR4” refers toan ADC preparation that has not been subjected to purification toisolate specific DAR peaks and comprises a heterogeneous mixture of ADCmolecules having different numbers of cytostatic and/or cytotoxic agentsattached per antibody (e.g., 0, 2, 4, 6, 8 agents per antibody), but hasan average drug-to-antibody ratio of 4. Similarly, “DAR8” refers to aheterogeneous ADC preparation in which the average drug-to-antibodyratio is 8.

Heterogeneous ADC preparations may be processed, for example, byhydrophobic interaction chromatography (“HIC”) to yield preparationsenriched in an ADC having a specified DAR of interest (or a mixture oftwo or more specified DARS). Such enriched preparations are designedherein as “EX,” where “E” indicates the ADC preparation has beenprocessed and is enriched in an ADC having a specific DAR and “X”represents the number of cytostatic and/or cytotoxic agents linked perADC molecule. Preparations enriched in a mixture of ADCs having twospecific DARs are designated “EX/EY,” three specific DARs “EX/EY/EZ”etc., where “E” indicates the ADC preparation has been processed toenrich the specified DARs and “X,” “Y” and “Z” represent the DARsenriched. As specific examples, “E2” refers to an ADC preparation thathas been enriched to contain primarily ADCs having two cytostatic and/orcytotoxic agents linked per ADC molecule. “E4” refers to an ADCpreparation that has been enriched to contain primarily ADCs having fourcytostatic and/or cytotoxic agents linked per ADC molecule. “E2/E4”refers to an ADC preparation that has been enriched to contain primarilytwo ADC populations, one having two cytostatic and/or cytotoxic agentslinked per ADC molecule and another having four cytostatic and/orcytotoxic agents linked per ADC molecule.

An enriched “E” preparation can also refer to an ADC that has beenprepared from an antibody that has been engineered, e.g., by insertionor deletion of a cysteine residue, to form a linkage to a drug at aspecific site. For example, an antibody with a S239C mutation in eachheavy chain can primarily have a drug attached via a linker at thatsite, and, hence, can afford an E2 ADC preparation having mostly DAR2without additional processing, such as chromatographic processing.

As used herein, enriched “E” preparations will generally be at leastabout 80% pure in the stated DAR ADCs, although higher levels of purity,such as purities of at least about 85%, 90%, 95%, 98%, 99% or evenhigher, may be obtainable and desirable. In some embodiments, theenriched “E” preparations have a range of purity within any two of theforegoing values, such as but not limited to from about 80-99%, 80-98%,85-95%, 90-98%, 95-98%, or 80-90%. For example, an “EX” preparation willgenerally be at least about 80% pure in ADCs having X cytostatic and/orcytotoxic agents linked per ADC molecule. For “higher order” enrichedpreparations, such as, for example, “EX/EY” preparations, the sum totalof ADCs having X and Y cytostatic and/or cytotoxic agents linked per ADCmolecule will generally comprise at least about 80% of the total ADCs inthe preparation. Similarly, in an enriched “EX/EY/EZ” preparation, thesum total of ADCs having X, Y and Z cytostatic and/or cytotoxic agentslinked per ADC molecule will comprise at least about 80% of the totalADCs in the preparation.

Purity may be assessed by a variety of methods, as is known in the art.As a specific example, an ADC preparation may be analyzed via HPLC orother chromatography and the purity assessed by analyzing areas underthe curves of the resultant peaks. Specific chromatography methods thatmay be employed to assess purity of ADC preparations are provided inExample 8.

FIG. 12 is illustrative. The top panel shows a chromatogram of a crudepreparation of an ADC prepared according to Example 8. The preparationcontains antibodies having no cytostatic and/or cytotoxic agentsattached (DAR0), two agents attached (DAR2), four agents (DAR4), sixagents attached (DAR6) and eight agents attached (DAR8). This crudepreparation has an average DAR of 4. HIC chromatography yields an E2preparation in which approximately 95% of the ADCs in the preparationhave two cytostatic and/or cytotoxic agents linked per ADC molecule(stated another way, approximately 95% of the ADCs are DAR2).

Specific methods for obtaining heterogenous mixtures of ADCs comprisinghumanized antibody huM25 having an average DAR of 4, as well as highlypurified preparations containing 2 and 4 linked agents are provided inthe Examples section. These specific methods may be modified usingroutine skill to obtain heterogeous and/or homogeneous ADCs comprisingother anti-huLRRC15 antibodies, linkers and/or cytotoxic and/orcytostatic agents.

7.6. Compositions

The ADCs described herein may be in the form of compositions comprisingthe ADC and one or more carriers, excipients and/or diluents. Thecompositions may be formulated for specific uses, such as for veterinaryuses or pharmaceutical uses in humans. The form of the composition(e.g., dry powder, liquid formulation, etc.) and the excipients,diluents and/or carriers used will depend upon the intended uses of theantibody and/or ADC and, for therapeutic uses, the mode ofadministration.

For therapeutic uses, the compositions may be supplied as part of asterile, pharmaceutical composition that includes a pharmaceuticallyacceptable carrier. This composition can be in any suitable form(depending upon the desired method of administering it to a patient).The pharmaceutical composition can be administered to a patient by avariety of routes such as orally, transdermally, subcutaneously,intranasally, intravenously, intramuscularly, intratumorally,intrathecally, topically or locally. The most suitable route foradministration in any given case will depend on the particular antibodyand/or ADC, the subject, and the nature and severity of the disease andthe physical condition of the subject. Typically, the pharmaceuticalcomposition will be administered intravenously or subcutaneously.

Pharmaceutical compositions can be conveniently presented in unit dosageforms containing a predetermined amount of an antibody and/or ADCdescribed herein per dose. The quantity of antibody and/or ADC includedin a unit dose will depend on the disease being treated, as well asother factors as are well known in the art. Such unit dosages may be inthe form of a lyophilized dry powder containing an amount of antibodyand/or ADC suitable for a single administration, or in the form of aliquid. Dry powder unit dosage forms may be packaged in a kit with asyringe, a suitable quantity of diluent and/or other components usefulfor administration. Unit dosages in liquid form may be convenientlysupplied in the form of a syringe pre-filled with a quantity of antibodyand/or ADC suitable for a single administration.

The pharmaceutical compositions may also be supplied in bulk formcontaining quantities of ADC suitable for multiple administrations.

Pharmaceutical compositions may be prepared for storage as lyophilizedformulations or aqueous solutions by mixing an antibody and/or ADChaving the desired degree of purity with optionalpharmaceutically-acceptable carriers, excipients or stabilizerstypically employed in the art (all of which are referred to herein as“carriers”), i.e., buffering agents, stabilizing agents, preservatives,isotonifiers, non-ionic detergents, antioxidants, and othermiscellaneous additives. See, Remington's Pharmaceutical Sciences, 16thedition (Osol, ed. 1980). Such additives should be nontoxic to therecipients at the dosages and concentrations employed.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They may be present at a wide variety ofconcentrations, but will typically be present in concentrations rangingfrom about 2 mM to about 50 mM. Suitable buffering agents for use withthe present disclosure include both organic and inorganic acids andsalts thereof such as citrate buffers (e.g., monosodium citrate-disodiumcitrate mixture, citric acid-trisodium citrate mixture, citricacid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumgluconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium gluconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additionally, phosphate buffers, histidinebuffers and trimethylamine salts such as Tris can be used.

Preservatives may be added to retard microbial growth, and can be addedin amounts ranging from about 0.2%-1% (w/v). Suitable preservatives foruse with the present disclosure include phenol, benzyl alcohol,meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzylammonium chloride, benzalconium halides (e.g., chloride, bromide, andiodide), hexamethonium chloride, and alkyl parabens such as methyl orpropyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.Isotonicifiers sometimes known as “stabilizers” can be added to ensureisotonicity of liquid compositions of the present disclosure and includepolyhydric sugar alcohols, for example trihydric or higher sugaralcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol andmannitol. Stabilizers refer to a broad category of excipients which canrange in function from a bulking agent to an additive which solubilizesthe therapeutic agent or helps to prevent denaturation or adherence tothe container wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinositol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglyceroland sodium thiosulfate; low molecular weight polypeptides (e.g.,peptides of 10 residues or fewer); proteins such as human serum albumin,bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers,such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose,fructose, glucose; disaccharides such as lactose, maltose, sucrose andtrehalose; and trisaccacharides such as raffinose; and polysaccharidessuch as dextran. Stabilizers may be present in amounts ranging from 0.5to 10 weight % per weight of ADC.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe added to help solubilize the glycoprotein as well as to protect theglycoprotein against agitation-induced aggregation, which also permitsthe formulation to be exposed to shear surface stressed without causingdenaturation of the protein. Suitable non-ionic surfactants includepolysorbates (20, 80, etc.), poloxamers (184, 188 etc.), and pluronicpolyols. Non-ionic surfactants may be present in a range of about 0.05mg/mL to about 1.0 mg/mL, for example about 0.07 mg/mL to about 0.2mg/mL.

Additional miscellaneous excipients include bulking agents (e.g.,starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbicacid, methionine, vitamin E), and cosolvents.

A specific exemplary embodiment of an aqueous composition suitable foradministration via intravenous infusion comprises 20 mg/mL anti-huLRRC15ADC, 10 mM histidine buffer, pH 6.0, 7% (w/v) sucrose, 0.03% (w/v)polysorbate 80. The composition may be in the form of a lyophilizedpowder that, upon reconstitution with 5.2 mL sterile water or othersolution suitable for injection or infusion (for example, 0.9% saline,Ringer's solution, lactated Ringer's solution, etc.) provides the aboveaqueous composition. This embodiment, or other embodiments ofcompositions, may also be in the form of a syringe or other devicesuitable for injection and/or infusion pre-filled with a quantity ofcomposition suitable for a single administration of anti-huLRRC15 ADC.

7.7. Methods of Use

As discussed previously, for a variety of solid tumors, huLRRC15 isexpressed in the tumor stromal microenvironment, but not on the cancercells per se. Data provided herein demonstrate that anti-huLRRC15 ADCsexert potent anti-tumor activity against these stromal(+)/cancer(−)tumors in vivo. Accordingly, the ADCs and/or pharmaceutical compositionscomprising the ADCs may be used therapeutically to treatstromal(+)/cancer(−) tumors.

Generally, the methods involve administering to a human patient having astromal(+)/cancer(−) tumor an amount of an anti-huLRRC15 ADC effectiveto provide therapeutic benefit. Stromal(+)/cancer(−) tumors that may betreated with the ADCs include, but are not limited to, adrenal cancers,bladder cancers, breast cancers (e.g., ductal breast cancer, lobularbreast cancer, triple negative breast cancer), cervical cancers,endometrial cancers, gastric cancers, lung cancers (for example,mesothelioma and non-small cell lung cancers such as non-small cell lungadenocarcinoma and squamous non-small cell lung cancer), head and neckcancers, liver cancers (e.g., hepatocellular carcinomas), lymphomas(e.g., non-Hodgkin's lymphomas such as mantle cell lymphoma, follicularlymphoma, diffuse large B cell lymphoma), pancreatic cancers, colorectalcancers, ovarian cancers, renal cancers, stomach cancers, testicularcancers, thyroid cancers, and uterine cancers. The cancer may be newlydiagnosed and naïve to treatment, or may be relapsed, refractory, orrelapsed and refractory, or a metastatic form of a huLRRC15stromal(+)/cancer(−) tumor.

Indeed, as demonstrated in FIGS. 20A-20E, anti-huLRRC15 ADCs are moreeffective than both non-targeted and targeted anti-cancer agents againsta variety of stromal(+)/cancer(−) tumors. As demonstrated in FIGS.20A-20E, stromal(+)/cancer(−) tumors that exhibit resistance to othertargeted or non-targeted chemotherapies, retain sensitivity toanti-huLRRC15 ADCs.

Moreover, as shown in FIG. 14, stromal(+)/cancer(−) tumors thateventually regrow following treatment with anti-huLRRC15 ADCs remainsensitive to retreatment with anti-huLRRC15 ADCs. Accordingly, theanti-huLRRC15 ADCs described herein provide significant benefits overcurrent targeted and non-targeted approaches toward the treatment ofhuLRRC15 stromal(+)/cancer(−) tumors.

While not wishing to be bound by theory, the anti-huLRRC15 ADCs mayexhibit an anti-tumor effect in part by killing cancer cells that haveundergone epithelial-mesenchymal transition (EMT) and have acquired stemcell-like properties such as a mesenchymal phenotype. Data providedherein indicated that cancer cells that acquired mesenchymal-likeproperties exhibited higher huLRRC15 expression than cells withepithelial characteristics (FIGS. 10 and 11A-11C). Cancer cells thathave undergone EMT transition were more sensitive to an anti-huLRRC15ADC than epithelial cancer cells (FIG. 23D). The higher killing effectmay result in part due to the localization of the ADC toLRRC15-expressing cells, followed by release of the cytotoxic agent.Accordingly, an anti-tumor effect exhibited by administration of ananti-huLRRC15 ADC (FIGS. 24A-24G) may be a result in part of thetargeting and killing of cancer cells that have undergone EMT andacquired cancer stem cell like properties.

FIGS. 9A-9C demonstrate that bone marrow derived mesenchymal stem cells,hypothesized to localize to the tumor microenvironment and form part ofthe tumor stroma, exhibited significant levels of huLRRC15 expression(Karnoub, A E et al., Nature (2007), 449, 557-563; Droujinine, I A etal., Oncotarget (2013), 4(5), 651-664). Additionally, significanthuLRRC15 expression can be induced by TGFβ in these mesenchymal stemcells (FIGS. 9A-9C). Bone marrow mesenchymal stem cells (BM-MSCs)stimulated with TGFβ to express significant levels of LRRC15 (FIGS. 23Band 23C), were sensitive to killing by an anti-huLRRC15 ADC. The killingeffect may be a result in part of the localization to LRRC15-expressingcells by the ADC, followed by release of the cytotoxic agent.Accordingly, an anti-tumor effect exhibited by administration of ananti-huLRRC15 ADC (FIGS. 24A-24G) may be a result in part of thetargeting and/or killing of mesenchymal stem cells that make up part ofthe fibroblast population in the tumor stromal microenvironment. Inaddition, FIGS. 23B and 23C demonstrate that mesenchymal stem cells areable to take up and process anti-huLRRC15 ADCs, which may directly killthe mesenchymal stem cell as well as kill the cancer cells in proximityto the mesenchymal stem cells via a bystander effect through the releaseof the cytotoxic and/or cytostatic agent.

Anti-huLRRC15 ADCs may be administered alone (monotherapy) or adjunctiveto, or with, other anti-cancer therapies and/or targeted or non-targetedanti-cancer agents. When administered as anti-huLRRC15 ADC monotherapy,one or more anti-huLRRC15 ADCs may be used. Whether administered asmonotherapy or adjunctive to, or with, other therapies or agents, anamount of anti-huLRRC15 ADC is administered such that the overalltreatment regimen provides therapeutic benefit.

By therapeutic benefit is meant that the use of anti-huLRRC15 ADCs totreat cancer in a patient results in a demonstrated improvement insurvival compared with no therapy (when appropriate) or to a knownstandard of care. In some cases, therapeutic benefit may constitute animprovement in time to disease progression together with an improvementin symptoms or quality of life. In other cases, therapeutic benefit maynot translate to an increased period of disease control, but rather amarkedly reduced symptom burden resulting in improved quality of life.As will be apparent to those of skill in the art, a therapeutic benefitmay be observed using the anti-huLRRC15 ADCs alone (monotherapy) oradjunctive to, or with, other anti-cancer therapies and/or targeted ornon-targeted anti-cancer agents.

Typically, therapeutic benefit is assessed using standard clinical testsdesigned to measure the response to a new treatment for cancer. Toassess the therapeutic benefits of the anti-huLRRC15 ADCs describedherein one or a combination of the following tests can be used: (1) theResponse Evaluation Criteria In Solid Tumors (RECIST) version 1.1, (2)immune-related RECIST (irRECIST), (3) the Eastern Cooperative OncologyGroup (ECOG) Performance Status, (4) immune-related response criteria(irRC), (5) disease evaluable by assessment of tumor antigens, (6)validated patient reported outcome scales, and/or (7) Kaplan-Meierestimates for overall survival and progression free survival.

Assessment of the change in tumor burden is an important feature of theclinical evaluation of cancer therapeutics. Both tumor shrinkage(objective response) and time to the development of disease progressionare important endpoints in cancer clinical trials. Standardized responsecriteria, known as RECIST (Response Evaluation Criteria in SolidTumors), were published in 2000. An update (RECIST 1.1) was released in2009. RECIST criteria are typically used in clinical trials whereobjective response is the primary study endpoint, as well as in trialswhere assessment of stable disease, tumor progression or time toprogression analyses are undertaken because these outcome measures arebased on an assessment of anatomical tumor burden and its change overthe course of the trial. TABLE 3 provides the definitions of theresponse criteria used to determine objective tumor response to a studydrug, such as the anti-huLRRC15 ADCs described herein.

TABLE 3 Response Criteria Complete Response Disappearance of all targetlesions. Any pathological lymph nodes (CR) (whether target ornon-target) must have reduction in short axis to <10 mm. PartialResponse At least a 30% decrease in the sum of diameters of targetlesions, taking as (PR) reference the baseline sum diameters.Progressive Disease At least a 20% increase in the sum of diameters oftarget lesions, taking as (PD) reference the smallest sum on study (thisincludes the baseline sum if that is the smallest on study). In additionto the relative increase of 20%, the sum must also demonstrate anabsolute increase of at least 5 mm. (Note: the appearance of one or morenew lesions is also considered progression). Stable Disease Neithersufficient shrinkage to qualify for PR nor sufficient increase to (SD)qualify for PD, taking as reference the smallest sum diameters while onstudy.

Secondary outcome measures that can be used to determine the therapeuticbenefit of the anti-huLRRC15 ADCs described herein include, ObjectiveResponse Rate (ORR), Progression Free Survival (PFS), Overall Survival(OS), Duration of Overall Response (DOR), and Depth of Response (DpR).ORR is defined as the proportion of the participants who achieve acomplete response (CR) or partial response (PR). PFS is defined as thetime from the first dose date of an anti-huLRRC15 ADC to either diseaseprogression or death, whichever occurs first. OS is defined as thelength of time from either the date of diagnosis or the start oftreatment for a disease, that patients diagnosed with the disease arestill alive. DOR is defined as the time from the participant's initialCR or PR to the time of disease progression. DpR is defined as thepercentage of tumor shrinkage observed at the maximal response pointcompared to baseline tumor load. Clinical endpoints for both ORR and PFScan be determined based on RECIST 1.1 criteria described above.

Additional criteria that may be used for clinical evaluation specific tocancer patients undergoing immune therapy treatment include thestandardized immune-related RECIST (irRECIST) criteria. See, e.g.,Nishino, M. et al. Eur. J. Radiol., 84(7), pages 1259-1268 (2015 July).These guidelines modified the RECIST 1.1 criteria above withconsideration of potential immunomodulatory effects. TABLE 5 providesthe definitions of the response criteria used to determine objectivetumor response to an immunomodulatory drug, such as the anti-huLRRC15ADCs described herein.

TABLE 4 Response Criteria Complete Response Complete disappearance ofall measurable and non-measurable lesions. (irCR) Lymph nodes mustdecrease to <10 mm in short axis. Partial Response Decrease of ≥30% intotal measured tumor burden relative to baseline, (irPR) non-targetlesions are irNN, and no unequivocal progression of new non- measurablelesions Progressive Disease At least a 20% increase and at least 5 mmabsolute increase in TMTB (irPD) compared to nadir, or irPD fornon-target or new non-measurable lesions. Confirmation of progression isrecommended at least 4 weeks after the first irPD assessment. Non-irCRor non- No target disease was identified at baseline and at follow-upthe patient irPD (irNN) fails to meet criteria for irCR or irPD StableDisease Neither sufficient shrinkage to qualify for irPR nor sufficientincrease to (irSD) qualify for irPD, taking as reference the smallestsum diameters while on study. irNE Used in exceptional cases whereinsufficient data exists.

The ECOG Scale of Performance Status shown in TABLE 5 is used todescribe a patient's level of functioning in terms of their ability tocare for themselves, daily activity, and physical ability. The scale wasdeveloped by the Eastern Cooperative Oncology Group (ECOG), now part ofthe ECOG-ACRIN Cancer Research Group, and published in 1982.

TABLE 5 Grade ECOG Performance Status 0 Fully active, able to carry onall pre-disease performance without restriction 1 Restricted inphysically strenuous activity but ambulatory and able to carry out workof a light or sedentary nature, e.g., light house work, office work 2Ambulatory and capable of all selfcare but unable to carry out any workactivities; up and about more than 50% of waking hours 3 Capable of onlylimited selfcare; confined to bed or chair more than 50% of waking hours4 Completely disabled; cannot carry on any selfcare; totally confined tobed or chair 5 Dead

Another set of criteria that can be used to characterize fully and todetermine response to immunotherapeutic agents, such as antibody-basedcancer therapies, is the immune-related response criteria (irRC), whichwas developed for measurement of solid tumors in 2009, and updated in2013 (Wolchok, et al. Clin. Cancer Res. 2009; 15(23): 7412-7420 andNishino, et al. Clin. Cancer Res. 2013; 19(14): 3936-3943, each of whichis incorporated by reference in its entirety). The updated irRC criteriaare typically used to assess the effect of an immunotherapeutic agent,such as an anti-huLRRC15 ADC described herein, on tumor burden, anddefines response according to TABLE 6.

TABLE 6 Response Criteria Complete Response Disappearance of all targetlesions in two consecutive observations not less (CR) than 4 weeks apartPartial Response At least a 30% decrease in the sum of the longestdiameters of target (PR) lesions, taking as reference the baseline sumdiameters. Progressive Disease At least a 20% increase in the sum ofdiameters of target lesions, taking as (PD) reference the smallest sumon study (this includes the baseline sum if that is the smallest onstudy). (Note: the appearance of one or more new lesions is notconsidered progression. The measurement of new lesions is included inthe sum of the measurements). Stable Disease Neither sufficientshrinkage to qualify for PR nor sufficient increase to (SD) qualify forPD, taking as reference the smallest sum diameters while on study.

Tumor antigens that can be used to evaluate the therapeutic benefit ofthe anti-huLRRC15 ADCs described herein include ApoE, CD11c, CD40, CD45(PTPRC), CD49D (ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1,PIK3CD, CD74, CCL5, CCR5, CXCL10, IFNG, IL10RA1, IL-6, ACTA2, COL7A1,LOX, LRRC15, MCPT8, MMP10, NOG, SERPINE1, STAT1, TGFBR1, CTSS, PGF,VEGFA, C1QA, C1QB, ANGPTL4, EGLN, ANGPTL4, EGLN3, BNIP3, AIF1, CCL5,CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1,AKR1C1, AKR1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A,IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CD11b, ADGRE1(EMR1, F4/80), CD86, CD68, MHC-Class II, CD3, HLA-DR, CD4, CD3, CD5,CD19, CD7, CD8, CD16, TCRαβ, TCRγδ, PD-1, PDL-1, CTLA-4, acidphosphatase, ACTH, alkaline phosphatase, alpha-fetoprotein CA-125,CA15-3, CA19-9, CA-195, C-212, CA-549, calcitonin, catecholamines,cathepsin-D, CEA, ERBB2 (HER2/neu), chromagranin-A, c-Myc, EGFR, ERA(estrogen receptor assay), ferritin, gastrin, 5-HIAA, hCG, alpha-HCG,beta-HCG, HVA, LDH1-5, NSE (neuron specific enolase), pancreaticpolypeptide, PLAP, PLP, PRA (progesterone receptor A), proinsulinC-peptide, PSA, SMA, SCC, thyroglobulin, TDT, TPA, and alpha-TSH. Theseantigens can be assessed at the DNA, RNA or protein level using DNAsequencing techniques, RNA sequencing techniques, gene chip microarray,PCR based methods, flow cytometry or immunohistochemistry methods asknown to experts in the art.

One exemplary therapeutic benefit resulting from the use ofanti-huLRRC15 ADCs described herein to treat stromal(+)/cancer(−)tumors, whether administered as monotherapy or adjunctive to, or with,other therapies or agents, is a complete response. Another exemplarytherapeutic benefit resulting from the use of anti-huLRRC15 ADCsdescribed herein to treat stromal(+)/cancer(−) tumors, whetheradministered as monotherapy or adjunctive to, or with, other therapiesor agents, is a partial response.

Validated patient reported outcome scales can also be used to denoteresponse provided by each patient through a specific reporting system.Rather than being disease focused, such outcome scales are concernedwith retained function while managing a chronic condition. Onenon-limiting example of a validated patient reported outcome scale isPROMIS® (Patient Reported Outcomes Measurement Information System) fromthe United States National Institutes of Health. For example, PROMIS®Physical Function Instrument for adult cancer patients can evaluateself-reported capabilities for the functioning of upper extremities(e.g., dexterity), lower extremities (e.g., walking or mobility), andcentral regions (e.g., neck, back mobility), and also includes routinedaily activities, such as running errands.

Kaplan-Meier curves (Kaplan and Meier, J. Am. Stat. Assoc. 1958;53(282): 457-481) can also be used to estimate overall survival andprogression free survival for cancer patients undergoing anti-huLRRC15antibody or ADC therapy in comparison to standard of care.

7.7.1. Adjunctive Therapies

Anti-huLRRC15 ADCs may be used adjunctive to, or with, other agents ortreatments having anti-cancer properties. When used adjunctively, theanti-huLRRC15 and other agent(s) may be formulated together in a single,combination pharmaceutical formulation, or may be formulated andadministered separately, either on a single coordinated dosing regimenor on different dosing regimens. Agents administered adjunctively withanti-huLRRC15 ADCs will typically have complementary activities to theanti-huLRRC15 ADCs such that the ADCs and other agents do not adverselyaffect each other.

Agents that may be used adjunctively with anti-huLRRC15 ADCs include,but are not limited to, alkylating agents, angiogenesis inhibitors,antibodies, antimetabolites, antimitotics, antiproliferatives,antivirals, aurora kinase inhibitors, apoptosis promoters (for example,Bcl-2 family inhibitors), activators of death receptor pathway, Bcr-Ablkinase inhibitors, BiTE (Bi-Specific T cell Engager) antibodies,antibody drug conjugates, biologic response modifiers, cyclin-dependentkinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors,DVDs, leukemia viral oncogene homolog (ErbB2) receptor inhibitors,growth factor inhibitors, heat shock protein (HSP)-90 inhibitors,histone deacetylase (HDAC) inhibitors, hormonal therapies,immunologicals, inhibitors of inhibitors of apoptosis proteins (IAPs),intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2inhibitors, mammalian target of rapamycin inhibitors, microRNAs,mitogen-activated extracellular signal-regulated kinase inhibitors,multivalent binding proteins, non-steroidal anti-inflammatory drugs(NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP)inhibitors, platinum chemotherapeutics, polo-like kinase (Plk)inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasomeinhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinaseinhibitors, retinoids/deltoids plant alkaloids, small inhibitoryribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligaseinhibitors, and the like, as well as combinations of one or more ofthese agents.

BiTE antibodies are bispecific antibodies that direct T-cells to attackcancer cells by simultaneously binding the two cells. The T-cell thenattacks the target cancer cell. Examples of BiTE antibodies includeadecatumumab (Micromet MT201), blinatumomab (Micromet MT103) and thelike. Without being limited by theory, one of the mechanisms by whichT-cells elicit apoptosis of the target cancer cell is by exocytosis ofcytolytic granule components, which include perforin and granzyme B.

SiRNAs are molecules having endogenous RNA bases or chemically modifiednucleotides. The modifications do not abolish cellular activity, butrather impart increased stability and/or increased cellular potency.Examples of chemical modifications include phosphorothioate groups,2′-deoxynucleotide, 2′-OCH3-containing ribonucleotides,2′-F-ribonucleotides, 2′-methoxyethyl ribonucleotides, combinationsthereof and the like. The siRNA can have varying lengths (e.g., 10-200bps) and structures (e.g., hairpins, single/double strands, bulges,nicks/gaps, mismatches) and are processed in cells to provide activegene silencing. A double-stranded siRNA (dsRNA) can have the same numberof nucleotides on each strand (blunt ends) or asymmetric ends(overhangs). The overhang of 1-2 nucleotides can be present on the senseand/or the antisense strand, as well as present on the 5′- and/or the3′-ends of a given strand.

Multivalent binding proteins are binding proteins comprising two or moreantigen binding sites. Multivalent binding proteins are engineered tohave the three or more antigen binding sites and are generally notnaturally occurring antibodies. The term “multispecific binding protein”means a binding protein capable of binding two or more related orunrelated targets. Dual variable domain (DVD) binding proteins aretetravalent or multivalent binding proteins binding proteins comprisingtwo or more antigen binding sites. Such DVDs may be monospecific (i.e.,capable of binding one antigen) or multispecific (i.e., capable ofbinding two or more antigens). DVD binding proteins comprising two heavychain DVD polypeptides and two light chain DVD polypeptides are referredto as DVD Ig's. Each half of a DVD Ig comprises a heavy chain DVDpolypeptide, a light chain DVD polypeptide, and two antigen bindingsites. Each binding site comprises a heavy chain variable domain and alight chain variable domain with a total of 6 CDRs involved in antigenbinding per antigen binding site.

Alkylating agents include, but are not limited to, altretamine, AMD-473,AP-5280, apaziquone, bendamustine, brostallicin, busulfan, carboquone,carmustine (BCNU), chlorambucil, CLORETAZINE® (laromustine, VNP 40101M),cyclophosphamide, dacarbazine, estramustine, fotemustine, glufosfamide,ifosfamide, KW-2170, lomustine (CCNU), mafosfamide, melphalan,mitobronitol, mitolactol, nimustine, nitrogen mustard N-oxide,ranimustine, temozolomide, thiotepa, TREANDA® (bendamustine),treosulfan, and trofosfamide.

Angiogenesis inhibitors include, but are not limited to,endothelial-specific receptor tyrosine kinase (Tie-2) inhibitors,epidermal growth factor receptor (EGFR) inhibitors, insulin growthfactor-2 receptor (IGFR-2) inhibitors, matrix metalloproteinase-2(MMP-2) inhibitors, matrix metalloproteinase-9 (MMP-9) inhibitors,platelet-derived growth factor receptor (PDGFR) inhibitors,thrombospondin analogs, and vascular endothelial growth factor receptortyrosine kinase (VEGFR) inhibitors.

Antimetabolites include, but are not limited to, ALIMTA® (pemetrexeddisodium, LY231514, MTA), 5-azacitidine, XELODA® (capecitabine),carmofur, LEUSTAT® (cladribine), clofarabine, cytarabine, cytarabineocfosfate, cytosine arabinoside, decitabine, deferoxamine,doxifluridine, eflornithine, EICAR(5-ethynyl-1-β-D-ribofuranosylimidazole-4-carboxamide), enocitabine,ethnylcytidine, fludarabine, 5-fluorouracil alone or in combination withleucovorin, GEMZAR® (gemcitabine), hydroxyurea, ALKERAN® (melphalan),mercaptopurine, 6-mercaptopurine riboside, methotrexate, mycophenolicacid, nelarabine, nolatrexed, ocfosfate, pelitrexol, pentostatin,raltitrexed, Ribavirin, triapine, trimetrexate, S-1, tiazofurin,tegafur, TS-1, vidarabine, and UFT.

Antivirals include, but are not limited to, ritonavir, acyclovir,cidofovir, ganciclovir, foscarnet, zidovudine, ribavirin, andhydroxychloroquine.

Aurora kinase inhibitors include, but are not limited to, ABT-348,AZD-1152, MLN-8054, VX-680, Aurora A-specific kinase inhibitors, AuroraB-specific kinase inhibitors and pan-Aurora kinase inhibitors.

Bcl-2 protein inhibitors include, but are not limited to, AT-101((−)gossypol), GENASENSE® (G3139 or oblimersen (Bcl-2-targetingantisense oligonucleotide)), IPI-194, IPI-565,N-(4-(4-((4′-chloro(1,1′-biphenyl)-2-yl)methyl)piperazin-1-yl)benzoyl)-4-(((1R)-3-(dimethylamino)-1-((phenylsulfanyl)methyl)propyl)amino)-3-nitrobenzenesulfonamide),N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohex-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)-4-(((1R)-3-(morpholin-4-yl)-1-((phenylsulfanyl)methyl)propyl)amino)-3-((trifluoromethyl)sulfonyl)benzenesulfonamide,venetoclax and GX-070 (obatoclax).

Bcr-Abl kinase inhibitors include, but are not limited to, DASATINIB®(BMS-354825) and GLEEVEC® (imatinib).

CDK inhibitors include, but are not limited to, AZD-5438, BMI-1040,BMS-032, BMS-387, CVT-2584, flavopyridol, GPC-286199, MCS-5A, PD0332991,PHA-690509, seliciclib (CYC-202, R-roscovitine), and ZK-304709.

COX-2 inhibitors include, but are not limited to, ABT-963, ARCOXIA®(etoricoxib), BEXTRA® (valdecoxib), BMS347070, CELEBREX® (celecoxib),COX-189 (lumiracoxib), CT-3, DERAMAXX® (deracoxib), JTE-522,4-methyl-2-(3,4-dimethylphenyl)-1-(4-sulfamoylphenyl-1H-pyrrole), MK-663(etoricoxib), NS-398, parecoxib, RS-57067, SC-58125, SD-8381, SVT-2016,S-2474, T-614, and VIOXX® (rofecoxib).

EGFR inhibitors include, but are not limited to, ABX-EGF, anti-EGFRimmunoliposomes, EGF-vaccine, EMD-7200, ERBITUX® (cetuximab), HR3, IgAantibodies, IRESSA® (gefitinib), TARCEVA® (erlotinib or OSI-774), TP-38,EGFR fusion protein, and TYKERB® (lapatinib).

ErbB2 receptor inhibitors include, but are not limited to, CP-724-714,CI-1033 (canertinib), HERCEPTIN® (trastuzumab), TYKERB® (lapatinib),OMNITARG® (2C4, pertuzumab), TAK-165, GW-572016 (ionafarnib), GW-282974,EKB-569, PI-166, dHER2 (HER2 vaccine), APC-8024 (HER-2 vaccine),anti-HER/2neu bispecific antibody, B7.her2IgG3, AS HER2 trifunctionalbispecific antibodies, mAB AR-209, and mAB 2B-1.

Histone deacetylase inhibitors include, but are not limited to,depsipeptide, LAQ-824, MS-275, trapoxin, suberoylanilide hydroxamic acid(SAHA), TSA, and valproic acid.

HSP-90 inhibitors include, but are not limited to, 17-AAG-nab, 17-AAG,CNF-101, CNF-1010, CNF-2024, 17-DMAG, geldanamycin, IPI-504, KOS-953,MYCOGRAB® (human recombinant antibody to HSP-90), NCS-683664, PU24FC1,PU-3, radicicol, SNX-2112, STA-9090, and VER49009.

Inhibitors of apoptosis proteins include, but are not limited to,HGS1029, GDC-0145, GDC-0152, LCL-161, and LBW-242.

Activators of death receptor pathway include, but are not limited to,TRAIL, antibodies or other agents that target TRAIL or death receptors(e.g., DR4 and DR5) such as Apomab, conatumumab, ETR2-ST01, GDC0145(lexatumumab), HGS-1029, LBY-135, PRO-1762 and trastuzumab.

Kinesin inhibitors include, but are not limited to, Eg5 inhibitors suchas AZD4877, ARRY-520; and CENPE inhibitors such as GSK923295A.

JAK-2 inhibitors include, but are not limited to, CEP-701 (lesaurtinib),XL019 and INCB018424.

MEK inhibitors include, but are not limited to, ARRY-142886,ARRY-438162, PD-325901, and PD-98059.

mTOR inhibitors include, but are not limited to, AP-23573, CCI-779,everolimus, RAD-001, rapamycin, temsirolimus, ATP-competitiveTORC1/TORC2 inhibitors, including PI-103, PP242, PP30, and Torin 1.

Non-steroidal anti-inflammatory drugs include, but are not limited to,AMIGESIC® (salsalate), DOLOBID® (diflunisal), MOTRIN® (ibuprofen),ORUDIS® (ketoprofen), RELAFEN® (nabumetone), FELDENE® (piroxicam),ibuprofen cream, ALEVE® (naproxen) and NAPROSYN® (naproxen), VOLTAREN®(diclofenac), INDOCIN® (indomethacin), CLINORIL® (sulindac), TOLECTIN®(tolmetin), LODINE® (etodolac), TORADOL® (ketorolac), and DAYPRO®(oxaprozin).

PDGFR inhibitors include, but are not limited to, C-451, CP-673 andCP-868596.

Platinum chemotherapeutics include, but are not limited to, cisplatin,ELOXATIN® (oxaliplatin) eptaplatin, lobaplatin, nedaplatin, PARAPLATIN®(carboplatin), satraplatin, and picoplatin.

Polo-like kinase inhibitors include, but are not limited to, BI-2536.

Phosphoinositide-3 kinase (PI3K) inhibitors include, but are not limitedto, wortmannin, LY294002, XL-147, CAL-120, ONC-21, AEZS-127, ETP-45658,PX-866, GDC-0941, BGT226, BEZ235, and XL765.

Thrombospondin analogs include, but are not limited to, ABT-510,ABT-567, ABT-898, and TSP-1.

VEGFR inhibitors include, but are not limited to, AVASTIN®(bevacizumab), ABT-869, AEE-788, ANGIOZYME™ (a ribozyme that inhibitsangiogenesis (Ribozyme Pharmaceuticals (Boulder, Colo.) and Chiron(Emeryville, Calif.)), axitinib (AG-13736), AZD-2171, CP-547,632,IM-862, MACUGEN® (pegaptamib), NEXAVAR® (sorafenib, BAY43-9006),pazopanib (GW-786034), vatalanib (PTK-787, ZK-222584), SUTENT®(sunitinib, SU-11248), VEGF trap, and ZACTIMA™ (vandetanib, ZD-6474).

Antibiotics include, but are not limited to, intercalating antibioticsaclarubicin, actinomycin D, amrubicin, annamycin, adriamycin, BLENOXANE®(bleomycin), daunorubicin, CAELYX® or MYOCET® (liposomal doxorubicin),elsamitrucin, epirbucin, glarbuicin, ZAVEDOS® (idarubicin), mitomycin C,nemorubicin, neocarzinostatin, peplomycin, pirarubicin, rebeccamycin,stimalamer, streptozocin, VALSTAR® (valrubicin), and zinostatin.

Topoisomerase inhibitors include, but are not limited to, aclarubicin,9-aminocamptothecin, amonafide, amsacrine, becatecarin, belotecan,BN-80915, CAMPTOSAR® (irinotecan hydrochloride), camptothecin,CARDIOXANE® (dexrazoxine), diflomotecan, edotecarin, ELLENCE® orPHARMORUBICIN® (epirubicin), etoposide, exatecan,10-hydroxycamptothecin, gimatecan, lurtotecan, mitoxantrone, Onivyde™(liposomal irinotecan), orathecin, pirarbucin, pixantrone, rubitecan,sobuzoxane, SN-38, tafluposide, and topotecan.

Antibodies include, but are not limited to, AVASTIN® (bevacizumab),CD40-specific antibodies, chTNT-1/B, denosumab, ERBITUX® (cetuximab),HUMAX-CD4® (zanolimumab), IGF1R-specific antibodies, lintuzumab,PANOREX® (edrecolomab), RENCAREX® (WX G250), RITUXAN® (rituximab),ticilimumab, trastuzumab, pertuzumab, VECTIBIX® (panitumumab) and CD20antibodies types I and II.

Hormonal therapies include, but are not limited to, ARIMIDEX®(anastrozole), AROMASIN® (exemestane), arzoxifene, CASODEX®(bicalutamide), CETROTIDE® (cetrorelix), degarelix, deslorelin, DESOPAN®(trilostane), dexamethasone, DROGENIL® (flutamide), EVISTA®(raloxifene), AFEMA™ (fadrozole), FARESTON® (toremifene), FASLODEX®(fulvestrant), FEMARA® (letrozole), formestane, glucocorticoids,HECTOROL® (doxercalciferol), RENAGEL® (sevelamer carbonate),lasofoxifene, leuprolide acetate, MEGACE® (megesterol), MIFEPREX®(mifepristone), NILANDRON™ (nilutamide), NOLVADEX® (tamoxifen citrate),PLENAXIS™ (abarelix), prednisone, PROPECIA® (finasteride), rilostane,SUPREFACT® (buserelin), TRELSTAR® (luteinizing hormone releasing hormone(LHRH)), VANTAS® (Histrelin implant), VETORYL® (trilostane ormodrastane), and ZOLADEX® (fosrelin, goserelin).

Deltoids and retinoids include, but are not limited to, seocalcitol(EB1089, CB1093), lexacalcitrol (KH1060), fenretinide, PANRETIN®(aliretinoin), ATRAGEN® (liposomal tretinoin), TARGRETIN® (bexarotene),and LGD-1550.

PARP inhibitors include, but are not limited to, ABT-888 (veliparib),olaparib, KU-59436, AZD-2281, AG-014699, BSI-201, BGP-15, INO-1001, andONO-2231.

Plant alkaloids include, but are not limited to, vincristine,vinblastine, vindesine, and vinorelbine.

Proteasome inhibitors include, but are not limited to, VELCADE®(bortezomib), KYPROLIS® (carfilzomib), MG132, NPI-0052, and PR-171.

Examples of immunologicals include, but are not limited to, interferons,immune checkpoint inhibitors, co-stimulatory agents, and otherimmune-enhancing agents. Interferons include interferon alpha,interferon alpha-2a, interferon alpha-2b, interferon beta, interferongamma-1a, ACTIMMUNE® (interferon gamma-1b) or interferon gamma-n1,combinations thereof and the like. Immune check point inhibitors includeantibodies that target PD-1 (e.g., pembrolizumab, nivolumab), PD-L1(e.g., durvalumab, atezolizumab, avelumab, MEDI4736, MSB0010718C andMPDL3280A), and CTLA4 (cytotoxic lymphocyte antigen 4; e.g., ipilimumab,tremelimumab). Additional exemplary anti-PD-1 antibodies include thosedescribed in U.S. provisional application No. 62/394,314, such as ananti-PD-1 antibody having a heavy chain amino acid sequence accordingto:

(SEQ ID NO: 91) EIQLVQSGAEVKKPGSSVKVSCKASGYTFTHYGMNWVRQAPGQGLEWVGWVNTYTGEPTYADDFKGRLTFTLDTSTSTAYMELSSLRSEDTAVYYCTREGEGLGFGDWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVIVHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK or (SEQ ID NO: 92)EIQLVQSGAEVKKPGSSVKVSCKASGYTFTHYGMNWVRQAPGQGLEWVGWVNTYTGEPTYADDFKGRLTFTLDTSTSTAYMELSSLRSEDTAVYYCTREGEGLGFGDWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSMHEALHNHYTQKSLSLSPG;anda light chain amino acid sequence according to:

(SEQ ID NO: 93) DVVMTQSPLSLPVTPGEPASISCRSSQSIVHSHGDTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPVTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC,wherein the underlined amino acids represent the CDRs and the italicizedamino acids represent the constant regions.

Co-stimulatory agents include, but are not limited to, antibodiesagainst CD3, CD40, CD40L, CD27, CD28, CSF1R, CD137 (e.g., urelumab),B7H1, GITR, ICOS, CD80, CD86, OX40, OX40L, CD70, HLA-DR, LIGHT, LIGHT-R,TIM3, A2AR, NKG2A, KIR (e.g., lirilumab), TGF-β (e.g., fresolimumab) andcombinations thereof.

Other agents include, but are not limited to, ALFAFERONE® (IFN-α),BAM-002 (oxidized glutathione), BEROMUN® (tasonermin), BEXXAR®(tositumomab), CAMPATH® (alemtuzumab), dacarbazine, denileukin,epratuzumab, GRANOCYTE® (lenograstim), lentinan, leukocyte alphainterferon, imiquimod, melanoma vaccine, mitumomab, molgramostim,MYLOTARG™ (gemtuzumab ozogamicin), NEUPOGEN® (filgrastim), OncoVAC-CL,OVAREX® (oregovomab), pemtumomab (Y-muHMFG1), PROVENGE® (sipuleucel-T),sargaramostim, sizofilan, teceleukin, THERACYS® (BacillusCalmette-Guerin), ubenimex, VIRULIZIN® (immunotherapeutic, LorusPharmaceuticals), Z-100 (Specific Substance of Maruyama (SSM)), WF-10(Tetrachlorodecaoxide (TCDO)), PROLEUKIN® (aldesleukin), ZADAXIN®(thymalfasin), ZINBRYTA® (daclizumab high-yield process), and ZEVALIN®(⁹⁰Y-Ibritumomab tiuxetan).

Biological response modifiers are agents that modify defense mechanismsof living organisms or biological responses, such as survival, growth ordifferentiation of tissue cells to direct them to have anti-tumoractivity and include, but are not limited to, krestin, lentinan,sizofiran, picibanil PF-3512676 (CpG-8954), and ubenimex.

Pyrimidine analogs include, but are not limited to, cytarabine (ara C orArabinoside C), cytosine arabinoside, doxifluridine, FLUDARA®(fludarabine), 5-FU (5-fluorouracil), floxuridine, GEMZAR®(gemcitabine), TOMUDEX® (ratitrexed), and TROXATYL™ (triacetyluridinetroxacitabine).

Purine analogs include, but are not limited to, LANVIS® (thioguanine)and PURI-NETHOL® (mercaptopurine).

Antimitotic agents include, but are not limited to, batabulin,epothilone D (KOS-862),N-(2-((4-hydroxyphenyl)amino)pyridin-3-yl)-4-methoxybenzenesulfonamide,ixabepilone (BMS 247550), TAXOL® (paclitaxel), TAXOTERE® (docetaxel),PNU100940 (109881), patupilone, XRP-9881 (larotaxel), vinflunine, andZK-EPO (synthetic epothilone).

Ubiquitin ligase inhibitors include, but are not limited to, MDM2inhibitors, such as nutlins, and NEDD8 inhibitors such as MLN4924.

Anti-huLRRC15 ADCs may also be used to enhance the efficacy of radiationtherapy. Examples of radiation therapy include external beam radiationtherapy, internal radiation therapy (i.e., brachytherapy) and systemicradiation therapy.

Anti-huLRRC15 ADCs may be administered adjunctive to or with otherchemotherapeutic agents such as ABRAXANE™ (ABI-007), ABT-100 (farnesyltransferase inhibitor), ADVEXIN® (Ad5CMV-p53 vaccine), ALTOCOR® orMEVACOR® (lovastatin), AMPLIGEN® (poly I:poly C12U, a synthetic RNA),APTOSYN® (exisulind), AREDIA® (pamidronic acid), arglabin,L-asparaginase, atamestane (1-methyl-3,17-dione-androsta-1,4-diene),AVAGE® (tazarotene), AVE-8062 (combreastatin derivative) BEC2(mitumomab), cachectin or cachexin (tumor necrosis factor), canvaxin(vaccine), CEAVAC® (cancer vaccine), CELEUK® (celmoleukin), CEPLENE®(histamine dihydrochloride), CERVARIX® (human papillomavirus vaccine),CHOP® (C: CYTOXAN® (cyclophosphamide); H: ADRIAMYCIN®(hydroxydoxorubicin); O: Vincristine (ONCOVIN®); P: prednisone), CYPAT™(cyproterone acetate), combrestatin A4P, DAB(389)EGF (catalytic andtranslocation domains of diphtheria toxin fused via a His-Ala linker tohuman epidermal growth factor) or TransMID-107R™ (diphtheria toxins),dacarbazine, dactinomycin, 5,6-dimethylxanthenone-4-acetic acid (DMXAA),eniluracil, EVIZON™ (squalamine lactate), DIMERICINE® (T4N5 liposomelotion), discodermolide, DX-8951f (exatecan mesylate), enzastaurin,EP0906 (epithilone B), GARDASIL® (quadrivalent human papillomavirus(Types 6, 11, 16, 18) recombinant vaccine), GASTRIMMUNE®, GENASENSE®,GMK (ganglioside conjugate vaccine), GVAX® (prostate cancer vaccine),halofuginone, histrelin, hydroxycarbamide, ibandronic acid, IGN-101,IL-13-PE38, IL-13-PE38QQR (cintredekin besudotox), IL-13-pseudomonasexotoxin, interferon-α, interferon-γ, JUNOVAN™ or MEPACT™ (mifamurtide),lonafarnib, 5,10-methylenetetrahydrofolate, miltefosine(hexadecylphosphocholine), NEOVASTAT® (AE-941), NEUTREXIN® (trimetrexateglucuronate), NIPENT® (pentostatin), ONCONASE® (a ribonuclease enzyme),ONCOPHAGE® (melanoma vaccine treatment), ONCOVAX® (IL-2 Vaccine),ORATHECIN™ (rubitecan), OSIDEM® (antibody-based cell drug), OVAREX® MAb(murine monoclonal antibody), paclitaxel, PANDIMEX™ (aglycone saponinsfrom ginseng comprising 20(S)protopanaxadiol (aPPD) and20(S)protopanaxatriol (aPPT)), panitumumab, PANVAC®-VF (investigationalcancer vaccine), pegaspargase, PEG Interferon A, phenoxodiol,procarbazine, rebimastat, REMOVAB® (catumaxomab), REVLIMID®(lenalidomide), RSR13 (efaproxiral), SOMATULINE® LA (lanreotide),SORIATANE® (acitretin), staurosporine (Streptomyces staurospores),talabostat (PT100), TARGRETIN® (bexarotene), TAXOPREXIN®(DHA-paclitaxel), TELCYTA® (canfosfamide, TLK286), temilifene, TEMODAR®(temozolomide), tesmilifene, thalidomide, THERATOPE® (STn-KLH), thymitaq(2-amino-3,4-dihydro-6-methyl-4-oxo-5-(4-pyridylthio)quinazolinedihydrochloride), TNFERADE™ (adenovector: DNA carrier containing thegene for tumor necrosis factor-α), TRACLEER® or ZAVESCA® (bosentan),tretinoin (Retin-A), tetrandrine, TRISENOX® (arsenic trioxide),VIRULIZIN®, ukrain (derivative of alkaloids from the greater celandineplant), vitaxin (anti-alphavbeta3 antibody), XCYTRIN® (motexafingadolinium), XINLAY™ (atrasentan), XYOTAX™ (paclitaxel poliglumex),YONDELIS® (trabectedin), ZD-6126, ZINECARD® (dexrazoxane), ZOMETA®(zolendronic acid), and zorubicin, as well as combinations of any ofthese agents.

7.8. Dosages and Administration Regimens

The amount of anti-huLRRC15 ADC administered will depend upon a varietyof factors, including but not limited to, the particular type ofstromal(+)/cancer(−) tumor treated, the stage of thestromal(+)/cancer(−) tumor being treated, the mode of administration,the frequency of administration, the desired therapeutic benefit, andother parameters such as the age, weight and other characteristics ofthe patient, etc. Determination of dosages effective to providetherapeutic benefit for specific modes and frequency of administrationis within the capabilities of those skilled in the art.

Dosages effective to provide therapeutic benefit may be estimatedinitially from in vivo animal models or clinical. Suitable animal modelsfor a wide variety of diseases are known in the art.

The anti-huLRRC15 ADCs may be administered by any route appropriate tothe condition to be treated. An anti-huLRRC15 ADC will typically beadministered parenterally, i.e., infusion, subcutaneous, intramuscular,intravenous (IV), intradermal, intrathecal, bolus, intratumor injectionor epidural ((Shire et al., 2004, J. Pharm. Sciences 93(6):1390-1402)).In one embodiment, an anti-huLRRC15 ADC is provided as a lyophilizedpowder in a vial. The vials may contain 50 mg, 100 mg, or 200 mg ofanti-huLRRC15 ADC. Prior to administration, the lyophilized powder isreconstituted with sterile water for injection (SWFI) or other suitablemedium to provide a solution containing 20 mg/mL anti-huLRRC15 ADC. Theresulting reconstituted solution is further diluted with saline or othersuitable medium and administered via an IV infusion once every 7 days,once every 14 days, once every 21 days, or once every 28 days. In someembodiments, for the first cycle, the infusion occurs over 180 minutes,subsequent infusions are over 90 minutes. In other embodiments, theinfusion occurs over 60 minutes.

In one exemplary embodiment, an anti-huLRRC15 ADC is administered onceevery 14 days at 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg,1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg,5.7 mg/kg, 6.0 mg/kg, 6.3 mg/kg, 6.6 mg/kg, 6.9 mg/kg, or 7.2 mg/kg.

In another exemplary embodiment, an anti-huLRRC15 ADC is administeredonce every 7 days at 0.15 mg/kg, 0.3 mg/kg, 0.45 mg/kg, 0.6 mg/kg, 0.9mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg,3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, or 4.2 mg/kg.

In another exemplary embodiment, an anti-huLRRC15 ADC is administeredonce every 28 days at 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg,3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4mg/kg, 5.7 mg/kg, 6.0 mg/kg, 6.3 mg/kg, 6.6 mg/kg, 6.9 mg/kg, 7.2 mg/kg,7.5 mg/kg, 7.8 mg/kg, 8.1 mg/kg, or 8.4 mg/kg.

In another exemplary embodiment, an anti-huLRRC15 ADC is administeredonce every 21 days at 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg,3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4mg/kg, 5.7 mg/kg, 6.0 mg/kg, 6.3 mg/kg, 6.6 mg/kg, 6.9 mg/kg, or 7.2mg/kg.

When administered adjunctive to, or with, other agents, such as otherchemotherapeutic agents, the ADCs may be administered on the sameschedule as the other agent(s), or on a different schedule. Whenadministered on the same schedule, the ADC may be administered before,after, or concurrently with the other agent. In some embodiments wherean ADC is administered adjunctive to, or with, standards of care, theADC may be initiated prior to commencement of the standard therapy, forexample a day, several days, a week, several weeks, a month, or evenseveral months before commencement of standard of care therapy.

In one exemplary embodiment, an anti-huLRRC15 ADC is used adjunctive togemcitabine (GEMZAR®) to treat pancreatic cancer. The anti-huLRRC15 ADCis administered via IV infusion once every 14 days at 0.3 mg/kg, 0.6mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg.Gemcitabine is administered by intravenous infusion at a dose of 1000mg/m² over 30 minutes once weekly for up to 7 weeks, followed by a weekof rest from treatment. If myelosuppression is observed, dosemodifications as provided in the prescribing information for gemcitabinemay be used. Subsequent cycles should consist of infusions once weeklyfor 3 consecutive weeks out of every 4 weeks. The adjunctiveanti-huLRRC15 ADC/gemcitabine therapy is continued until diseaseprogression or no longer tolerated by the patient.

In another exemplary embodiment, an anti-huLRRC15 ADC is used adjunctiveto paclitaxel albumin-stabilized nanoparticle formulation (ABRAXANE®) totreat pancreatic cancer. The anti-huLRRC15 ADC is administered via IVinfusion once every 14 days at 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg,3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg. The recommended dose andschedule for paclitaxel albumin-stabilized nanoparticle formulation is125 mg/m² administered as an intravenous infusion over 30-40 minutes ondays 1, 8, and 15 of each 28-day cycle. The adjunctive anti-huLRRC15ADC/ABRAXANE® therapy is continued until disease progression or nolonger tolerated by the patient.

In another exemplary embodiment, an anti-huLRRC15 ADC is used adjunctiveto paclitaxel albumin-stabilized nanoparticle formulation (ABRAXANE®)plus gemcitabine (GEMZAR®) to treat pancreatic cancer. The anti-huLRRC15ADC is administered via IV infusion once every 14 days at 0.3 mg/kg, 0.6mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg. Therecommended dose and schedule for paclitaxel albumin-stabilizednanoparticle formulation is 125 mg/m² administered as an intravenousinfusion over 30-40 minutes on days 1, 8, and 15 of each 28-day cycle.Gemcitabine is administered by intravenous infusion at a dose of 1000mg/m² over 30 minutes once weekly for up to 7 weeks (or until toxicityreducing or holding a dose), followed by a week of rest from treatment.Subsequent cycles should consist of infusions once weekly for 3consecutive weeks out of every 4 weeks. The adjunctive anti-huLRRC15ADC/ABRAXANE®/GEMZAR® therapy is continued until disease progression orno longer tolerated by the patient.

In yet another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to TARCEVA® (erlotinib) to treat pancreatic cancer. Theanti-huLRRC15 ADC is administered via IV infusion once every 14 days at0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg,4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0mg/kg. The recommended dose and schedule for erlotinib is 100 mg orally,once daily. The adjunctive anti-huLRRC15 ADC/erlotinib therapy iscontinued until disease progression or no longer tolerated by thepatient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to FOLFIRINOX to treat pancreatic cancer. The anti-huLRRC15ADC is administered via IV infusion once every 14 days at 0.3 mg/kg, 0.6mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg.FOLFIRINOX is a combination of four chemotherapy agents: fluorouracil[5-FU], leucovorin, irinotecan and oxaliplatin. In some embodiments,FOLFIRINOX is administered as follows: oxaliplatin, 85 mg/m²;irinotecan, 180 mg/m²; leucovorin, 400 mg/m²; and fluorouracil, 400mg/m² given as a bolus followed by 2400 mg/m² given as a 46-hourcontinuous infusion, every 2 weeks. The adjunctive anti-huLRRC15ADC/FOLFIRINOX therapy is continued until disease progression or nolonger tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to Onivyde® to treat pancreatic cancer. The anti-huLRRC15 ADCis administered via IV infusion once every 14 days at 0.3 mg/kg, 0.6mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg. Onivyde®is a liposomal irinotecan formulation. In some embodiments, Onivyde® isadministered at 70 mg/m² by intravenous infusion over 90 minutes every 2weeks. The adjunctive anti-huLRRC15 ADC/Onivyde® therapy is continueduntil disease progression or no longer tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to Onivyde®, fluorouracil, and leucovorin to treat pancreaticcancer. The anti-huLRRC15 ADC is administered via IV infusion once every14 days at 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg,3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7mg/kg or 6.0 mg/kg. Onivyde® is a liposomal irinotecan formulation. Insome embodiments, Onivyde® is administered at 70 mg/m² by intravenousinfusion over 90 minutes every 2 weeks, with leucovorin 400 mg/m² andfluorouracil 2400 mg/m² over 46 hours every 2 weeks. The adjunctiveanti-huLRRC15 ADC/Onivyde®/leucovorin/fluorouracil therapy is continueduntil disease progression or no longer tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to nivolumab (OPDIV®) to treat pancreatic cancer. Theanti-huLRRC15 ADC is administered via IV infusion once every 14 days at0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg,4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0mg/kg. Nivolumab is administered an intravenous infusion at 3 mg/kg over60 minutes every two weeks. The adjunctive anti-huLRRC15 ADC/nivolumabtherapy is continued until disease progression or no longer tolerated bythe patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC can be usedadjunctive to pembrolizumab (KEYTRUDA®) to treat pancreatic cancer. Theanti-huLRRC15 ADC is administered via IV infusion once every 21 days at0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg,4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0mg/kg. Pembrolizumab is administered as an intravenous infusion at 2mg/kg over 30 minutes every 3 weeks. The adjunctive anti-huLRRC15ADC/pembrolizumab therapy is continued until disease progression or nolonger tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to TARCEVA® (erlotinib) to treat non small cell lung cancer(NSCLC). The anti-huLRRC15 ADC is administered via IV infusion onceevery 14 days at 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg,1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg,5.7 mg/kg or 6.0 mg/kg. The recommended dose and schedule for erlotinibis 150 mg orally, once daily. The adjunctive anti-huLRRC15 ADC/erlotinibtherapy is continued until disease progression or no longer tolerated bythe patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to IRESSA® (gefitinib) to treat non small cell lung cancer(NSCLC). The anti-huLRRC15 ADC is administered via IV infusion onceevery 14 days at 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg,1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg,5.7 mg/kg or 6.0 mg/kg. The recommended dose and schedule for gefitinibis 250 mg orally, once daily. The adjunctive anti-huLRRC15 ADC/gefitinibtherapy is continued until disease progression or no longer tolerated bythe patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to afatinib to treat non small cell lung cancer (NSCLC). Theanti-huLRRC15 ADC is administered via IV infusion once every 14 days at0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg,4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0mg/kg. The recommended dose and schedule for afatinib is 40 mg orally,once daily. The adjunctive anti-huLRRC15 ADC/afatinib therapy iscontinued until disease progression or no longer tolerated by thepatient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to OPDIVO® (nivolumab) to treat non small cell lung cancer(NSCLC). The anti-huLRRC15 ADC is administered via IV infusion onceevery 14 days at 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg,1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg,5.7 mg/kg or 6.0 mg/kg. Nivolumab is administered an intravenousinfusion at 3 mg/kg over 60 minutes every two weeks. The adjunctiveanti-huLRRC15 ADC/nivolumab treatment is continued until diseaseprogression or no longer tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to OPDIVO® (nivolumab) and YERVOY® (ipilimumab) to treat nonsmall cell lung cancer (NSCLC). The anti-huLRRC15 ADC is administeredvia IV infusion once every 21 days at 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg,1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg,5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg, for four doses withipilimumab, then every 14 days at 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg,3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4mg/kg, 5.7 mg/kg or 6.0 mg/kg, without ipilimumab. Nivolumab isadministered as an intravenous infusion at 3 mg/kg over 60 minutes everytwo weeks. Ipilimumab is administered intravenously at 3 mg/kg over 90minutes every three weeks in the first four doses. The adjunctiveanti-huLRRC15 ADC/nivolumab/ipilimumab treatment is continued untildisease progression or no longer tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC can be usedadjunctive to pembrolizumab (KEYTRUDA®) to treat NSCLC. Theanti-huLRRC15 ADC is administered via IV infusion once every 21 days at0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.4mg/kg, 3.0 mg/kg, 3.6 mg/kg, 4.2 mg/kg, 4.8 mg/kg, 5.4 mg/kg, or 6.0mg/kg. Pembrolizumab is administered as an intravenous infusion at 2mg/kg over 30 minutes every 3 weeks. The adjunctive anti-huLRRC15 ADCand pembrolizumab therapy is continued until disease progression or nolonger tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to cisplatin to treat NSCLC. The anti-huLRRC15 ADC isadministered via IV infusion once every 14 days at 0.3 mg/kg, 0.6 mg/kg,0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg,4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg. Cisplatin isadministered at 20 mg/m² or more, once every 3 to 4 weeks. Theadjunctive anti-huLRRC15 ADC/cisplatin therapy is continued untildisease progression or no longer tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to carboplatin to treat NSCLC. The anti-huLRRC15 ADC isadministered via IV infusion once every 14 days at 0.3 mg/kg, 0.6 mg/kg,0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg,4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg. Carboplatin isadministered at 300 mg/m² or more, once every 4 weeks. The adjunctiveanti-huLRRC15 ADC/carboplatin therapy is continued until diseaseprogression or no longer tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to veliparib to treat NSCLC. The anti-huLRRC15 ADC isadministered via IV infusion once every 14 days at 0.3 mg/kg, 0.6 mg/kg,0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg,4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg. Veliparib isadministered orally, twice a day. The adjunctive anti-huLRRC15ADC/veliparib therapy is continued until disease progression or nolonger tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to veliparib and pemetrexed to treat NSCLC. The anti-huLRRC15ADC is administered via IV infusion once every 21 days at 0.3 mg/kg, 0.6mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg.Veliparib is administered orally, twice a day. Pemetrexed isadministered at 500 mg/m² intravenously every 21 days. The adjunctiveanti-huLRRC15 ADC/veliparib/pemetrexed therapy is continued untildisease progression or no longer tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to cetuximab to treat NSCLC. The anti-huLRRC15 ADC isadministered via IV infusion once every 14 days at 0.3 mg/kg, 0.6 mg/kg,0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg,4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg. Cetuximab isadministered at an initial dose of 400 mg/m² over a 120-minuteintravenous infusion followed by 250 mg/m² weekly infusion over 60minutes. The adjunctive anti-huLRRC15 ADC/cetuximab therapy is continueduntil disease progression or no longer tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to ipilimumab (YERVOY®) to treat NSCLC. The anti-huLRRC15 ADCis administered via IV infusion once every 21 days at 0.3 mg/kg, 0.6mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.4 mg/kg, 3.0 mg/kg,3.6 mg/kg, 4.2 mg/kg, 4.8 mg/kg, 5.4 mg/kg, or 6.0 mg/kg. Ipilimumab isadministered at 3 mg/kg intravenously over 90 minutes every 3 weeks for3 months. The anti-huLRRC15 ADC therapy is continued until diseaseprogression or no longer tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to radiation to treat NSCLC. The anti-huLRRC15 ADC isadministered via IV infusion once every 14 days at 0.3 mg/kg, 0.6 mg/kg,0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg, 2.7mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5 mg/kg,4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg. Typically,external beam radiation therapy is applied for a few minutes up to 5days a week for 5 to 7 weeks, but this will vary depending on the typeof external beam radiation therapy that is used. The adjunctiveanti-huLRRC15 ADC/radiation therapy is continued until diseaseprogression or no longer tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to doxorubicin to treat breast cancer. The anti-huLRRC15 ADCis administered via IV infusion once every 14 days at 0.3 mg/kg, 0.6mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg. Whenused adjunctively with other drugs, the most commonly used dosage ofdoxorubicin is 40 to 60 mg/m² given as a single intravenous injectionevery 21 to 28 days. The adjunctive anti-huLRRC15 ADC/doxorubicintherapy is continued until disease progression or no longer tolerated bythe patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to gemcitabine to treat breast cancer. The anti-huLRRC15 ADCis administered via IV infusion once every 14 days at 0.3 mg/kg, 0.6mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg.Gemcitabine is administered by intravenous infusion at a dose of 1000mg/m² over 30 minutes once weekly for up to 7 weeks (or until toxicityreducing or holding a dose), followed by a week of rest from treatment.Subsequent cycles should consist of infusions once weekly for 3consecutive weeks out of every 4 weeks. The adjunctive anti-huLRRC15ADC/gemcitabine therapy is continued until disease progression or nolonger tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to trastuzumab (HERCEPTIN®) to treat breast cancer. Theanti-huLRRC15 ADC is administered via IV infusion once every 14 days at0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg,4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0mg/kg. The recommended initial loading dose for trastuzumab is 4 mg/kgadministered as a 90-minute infusion. The recommended weekly maintenancedose for trastuzumab is 2 mg/kg which can be administered as a 30 minuteinfusion if the initial loading dose was well tolerated. The adjunctiveanti-huLRRC15 ADC/trastuzumab therapy is continued until diseaseprogression or no longer tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to capecitabine (XELODA®) to treat breast cancer. Theanti-huLRRC15 ADC is administered via IV infusion once every 21 days at0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg,4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0mg/kg. Capecitabine can be administered at 1250 mg/m² twice daily for 2weeks followed by a one week rest period in 3 week cycles. Theadjunctive anti-huLRRC15 ADC/capecitabine therapy is continued untildisease progression or no longer tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to nivolumab (OPDIVO®) to treat breast cancer. Theanti-huLRRC15 ADC is administered via IV infusion once every 14 days at0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg,4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0mg/kg. Nivolumab is administered an intravenous infusion at 3 mg/kg over60 minutes every two weeks. The adjunctive anti-huLRRC15 ADC/nivolumabtherapy is continued until disease progression or no longer tolerated bythe patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC can be usedadjunctive to pembrolizumab (KEYTRUDA®) to treat breast cancer. Theanti-huLRRC15 ADC is administered via IV infusion once every 21 days at0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg,4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0mg/kg. Pembrolizumab is administered as an intravenous infusion at 2mg/kg over 30 minutes every 3 weeks. The adjunctive anti-huLRRC15ADC/pembrolizumab therapy is continued until disease progression or nolonger tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctively to radiation to treat Head and Neck cancer. Theanti-huLRRC15 ADC is administered via IV infusion once every 14 days at0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg,4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0mg/kg. Typically, external beam radiation therapy is applied for a fewminutes up to 5 days a week for 5 to 7 weeks, but this will varydepending on the type of external beam radiation therapy that is used.The adjunctive anti-huLRRC15 ADC/radiation therapy is continued untildisease progression or no longer tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to cetuximab to treat Head and Neck cancer. The anti-huLRRC15ADC is administered via IV infusion once every 14 days at 0.3 mg/kg, 0.6mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg.Cetuximab is administered at an initial dose of 400 mg/m² over a120-minute intravenous infusion followed by 250 mg/m² weekly infusionover 60 minutes. The adjunctive anti-huLRRC15 ADC/cetuximab therapy iscontinued until disease progression or no longer tolerated by thepatient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to carboplatin to treat Head and Neck cancer. Theanti-huLRRC15 ADC is administered via IV infusion once every 14 days at0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg,4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0mg/kg. Carboplatin is administered at 300 mg/m² or more, once every 4weeks. The adjunctive anti-huLRRC15 ADC/carboplatin therapy is continueduntil disease progression or no longer tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to nivolumab (OPDIVO®) to treat Head and Neck cancer. Theanti-huLRRC15 ADC is administered via IV infusion once every 14 days at0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg,4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0mg/kg. Nivolumab is administered an intravenous infusion at 3 mg/kg over60 minutes every two weeks. The adjunctive anti-huLRRC15 ADC/nivolumabtherapy is continued until disease progression or no longer tolerated bythe patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC can be usedadjunctive to pembrolizumab (KEYTRUDA®) to treat Head and Neck cancer.The anti-huLRRC15 ADC is administered via IV infusion once every 21 daysat 0.3 mg/kg, 0.6 mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1mg/kg, 2.4 mg/kg, 2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg,4.2 mg/kg, 4.5 mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0mg/kg. Pembrolizumab is administered as an intravenous infusion at 2mg/kg over 30 minutes every 3 weeks. The adjunctive anti-huLRRC15ADC/pembrolizumab therapy is continued until disease progression or nolonger tolerated by the patient.

In still another exemplary embodiment, an anti-huLRRC15 ADC is usedadjunctive to cisplatin to treat Head and Neck cancer. The anti-huLRRC15ADC is administered via IV infusion once every 14 days at 0.3 mg/kg, 0.6mg/kg, 0.9 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2.1 mg/kg, 2.4 mg/kg,2.7 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.6 mg/kg, 3.9 mg/kg, 4.2 mg/kg, 4.5mg/kg, 4.8 mg/kg, 5.1 mg/kg, 5.4 mg/kg, 5.7 mg/kg or 6.0 mg/kg.Cisplatin is administered as an intravenous infusion at 100 mg/kg every3 weeks. The adjunctive anti-huLRRC15 ADC/cisplatin therapy is continueduntil disease progression or no longer tolerated by the patient.

As will be appreciated by those of skill in the art, the recommendeddosages for the various agents described above may need to be adjustedto optimize patient response and maximize therapeutic benefit.

8. EXAMPLES

The following Examples, which highlight certain features and propertiesof exemplary embodiments of anti-huLRRC15 ADCs are provided for purposesof illustration, and not limitation.

Example 1. Preparation of Exemplary Anti-huLRRC15 Antibodies

Antibodies against huLRRC15 were prepared against a cell line expressinghuLRRC15 (U118MG glioblastoma cells) using standard techniques.Exemplary antibodies having specified affinities and other desirablecharacteristics, for example, cross-reactive with cynomolgus LRRC15(“cynoLRRC15”), were isolated and certain of these antibodies humanized.Exemplary humanized antibodies include huM25, huAD208.4.1, huAD208.12.1,huAD208.14.1, hu139.1 and exemplary murine antibodies includemuAD209.9.1 and muAD210.40.9. Sequences of the V_(H) and V_(L) chains ofthese exemplary antibodies are provided in FIGS. 2A and 2B,respectively. Sequences of the heavy and light chains of exemplaryantibody huM25 are provided in FIGS. 3A and 3B, respectively. Theencoded heavy chain of huM25 is SEQ ID NO:18, and can be C-terminaltruncated upon expression in CHO cells to SEQ ID NO:102, with theencoded light chain of huM25 being SEQ ID NO:19.

Binding of these exemplary antibodies to endogenous huLRRC15, as well astheir respective EC₅₀s, was demonstrated with U118MG glioblastoma cellsvia a conventional flow cytometry assay using test antibodyconcentrations of 0.0001 μg/mL, 0.001 μg/mL, 0.01 μg/mL, 0.1 μg/mL, 1μg/mL, 10 μg/mL and 100 μg/mL. Isotype control antibodies (mouse orhuman) were used as appropriate. Flow cytometry binding data for variousrepresentative anti-huLRRC15 antibodies are shown in FIG. 4. TABLE 7provides EC₅₀ (nM) values for various anti-huLRRC15 antibodies asdetermined by flow cytometry.

TABLE 7 Binding of Antibodies to Cells Expressing huLRRC15 (FlowCytometry) U118MG U118MG Antibody EC₅₀ (nM) Antibody EC₅₀ (nM)muIgG_(2a) Isotype mAb huIgG₁ Isotype mAb NA muM25 0.496 huM25 0.498mu139.10 0.207 hu139.10 0.28 muAD208.4.1 0.934 huAD208.4.1 0.29muAD208.14.1 2.05 huAD208.14.1 5.25 muAD209.9.1 0.787 muAD210.40.9 0.08

Example 2. The Anti-huLRRC15 Antibodies Bind the Extracellular Domain ofhuLRRC15 Shed from the Cell Surface

Binding of the exemplary anti-huLRRC15 to the portion of theextracellular domain of huLRRC15 shed from the cell surface wasdemonstrated in an ELISA assay. huLRRC15-Fc fusion protein was generatedusing amino acid residues 22 to 526 of SEQ ID NO:3, which corresponds toa portion of the huLRRC15 extracellular protein domain to the cleavagesite. 96-well Immulon 4HBX plates (Thermo, cat. #3855) were coated with100 μL/well of this huLRRC15 fusion ECD protein at 2 μg/mL incarbonate-bicarbonate buffer (Thermo, cat.#28382) pH 9.4 and allowed tobind overnight at 4° C. Plates were washed three times with PBST andthen incubated with various concentrations of antibodies in PBST+0.3%BSA at room temperature for one hour. Plates were washed three timeswith PBST and then incubated with 100 μL of goat anti-human kappa lightchain HRP (Bethyl, cat.# A80-115P) at 1:5000 dilution for 30 min at RT.Plates were washed three times in PBST and 100 μL of TMB (SurmodicsBioFx cat.# TMBW-1000-01) was added to each well and incubated at RTuntil color developed (approximately 10 minutes). Reactions were stoppedby the addition of 650 nm Stop Reagent for TMB (Surmodics BioFx, cat.#BSTP-0100-01), and optical density (OD) was read at 650 nm (MolecularDevices Versamax PLUS). ELISA binding data for certain exemplaryanti-huLRRC15 antibodies are shown in FIG. 5. EC₅₀ values for variousanti-huLRRC15 antibodies are provided in TABLE 8, below.

All antibodies tested bound the huLRRC15 fusion with EC₅₀s in thesub-nanomolar range, indicating that the antibodies bind the portion ofthe huLRRC15 extracellular domain shed from the cell surface followingcleavage.

TABLE 8 Binding of Antibodies to huLRRC15 ECD (ELISA) huLRRC15-huLRRC15- ECD-Fc ECD-Fc Antibody EC₅₀ (nM) Antibody EC₅₀ (nM) muIgG_(2a)Isotype mAb NA huIgG₁ Isotype mAb NA muM25 0.22 huM25 0.26 mu139.10 0.15hu139.10 0.11 muAD208.4.1 0.15 huAD208.4.1 0.25 muAD208.14.1 0.22huAD208.14.1 0.8 muAD209.9.1 0.14

Example 3. Exemplary Anti-huLRRC15 Antibodies Bind to Different Epitopes

The ability of various exemplary anti-huLRRC15 antibodies to competewith muM25 for binding cells expressing huLRRC15, and hence whether theantibodies bind the same or different epitopes, was assessed in a flowcytometry competition assay using fluorescently labeled muM25(“muM25-AF488”) as a “reference antibody” and unlabeled anti-huLRRC15antibody as a “test antibody.” For the assay, aliquots of U118MG cells(200,000 cells per well) were incubated simultaneously with 1 μg/mLlabeled muM25 and either 0.0001, 0.001, 0.01, 0.1, 1, 10 or 100 μg/mLunlabeled test antibody and the amount of bound antibody (normalized toa control aliquot incubated with 1 μg/mL labeled muM25 alone) determinedvia flow cytometry. Isotype control antibodies (human or mouse) wereused as negative controls, and unlabeled muM25 was used as a positivecontrol.

In this assay, competition due to a test antibody binding to the same ora proximal epitope as the labeled reference antibody reduce binding ofthe reference labeled antibody. A positive result in this assay occurswhen a test antibody inhibits ≥20% of the binding of the fluorescentlylabeled reference antibody at a concentration of test antibody that is10 times greater than the concentration of the reference antibody.

Unlabeled huM25 competes fully with labeled muM25 as expected (FIG. 6A).Murine antibodies muAD208.4.1 and muAD208.14.1 partially compete withhuM25 (FIG. 6B). Both of these antibodies inhibit >20% of the binding oflabeled muM25. This indicates that they bind a similar or proximalhuLRRC15 epitope as muM25. In contrast, mu139.10, muAD208.12.1 andmuAD209.9.1 do not inhibit the binding of labeled muM25, demonstratingthat they bind distinct epitopes of huLRRC15 (FIG. 6B).

Example 4. huLRRC15 is Highly Expressed in Stroma of Major Solid TumorTypes

Expression of huLRRC15 in the stroma of various solid tumor types wasassessed using immunohistochemistry (IHC) staining on formalin fixedparaffin embedded (FFPE) tissues. Biopsies from different tumor typeswere used to generate tissue microarrays (TMA) which were assessed forhuLRRC15 expression. Tissue sections (4 μm) were cut, deparaffinized andantigen retrieval was performed using BORG Decloaker antigen retrievalbuffer at 125° C. for 1 min. Leica autostainer was used to block slidesand incubate with anti-huLRRC15 antibody (muAD210.40.9 at 1 μg/mL for 60minutes) and HRP anti-mouse secondary (Dako) together with DAB reagent(Dako) for detection. Results of the experiment are shown in TABLE 9 andFIG. 7.

The TMA samples were scored on a scale of 0 to 4. A score of ≥2 waschosen to identify tumors that express huLRRC15 at high levels. ThehuLRRC15 staining and expression data in TABLE 9 represents that seen inhuLRRC15 stromal positive, cancer negative tumors (FIG. 7).

A number of cancers, such as breast cancer (ductal and lobular as wellas triple negative) and head and neck cancer, showed uniformly strongexpression of huLRRC15 in tumor stroma, suggesting a broadly applicabletreatment regimen could be developed by targeting huLRRC15 in thosecancers. Other types of cancers that showed huLRRC15 positive expressionin some but not all tumor stromal samples included lung cancers (such asnon small cell lung cancers (NSCLC), e.g., adenocarcinoma and squamousNSCLC), pancreatic cancers, bladder cancers, hepatocellular carcinomas,colorectal cancers, ovarian cancers, non-Hodgkin's lymphoma (NHL) (suchas mantle cell lymphoma, follicular lymphoma, diffuse large B celllymphoma, and other lymphomas), testicular cancers, gastric cancers,endometrial cancers, and renal cancers. Prostate cancers did not showhuLRRC15 positive expression in stromal samples tested (0/25 samples).

TABLE 9 huLRRC15 Stromal Positive Expression in Cancer (detected byimmunohistochemistry) IHC Score (Stromal Expression) (TMA+ individualtissues) Tumor Type >2+ % Positive Breast Ductal + Lobular 72/76 95Triple Negative 10/11 91 Lung NSCLC - Adeno 63/87 72 NSCLC - Squamous 74/115 64 Head & Neck (incl. metastases) 47/53 89 Pancreatic 27/41 66Bladder 17/30 57 Hepatocellular  8/16 50 Colorectal 19/43 44 Ovarian 8/18 44 Total 22/51 43 Mantle cell lymphoma 4/5 80 NHL Follicularlymphoma  9/15 60 Diffuse large B cell 3/6 50 Other NHL  6/25 25Testicular 11/35 31 Gastric  2/23 9 Endometrial  2/30 7 Renal  1/30 3Prostate  0/25 0

Example 5. huLRRC15 Exhibits Limited Expression in Normal Tissues

The expression of huLRRC15 on normal, healthy tissues was assessed usingprotein immunohistochemical staining of normal tissues. The results areshown in FIG. 8. huLRRC15 has limited expression in most normal tissues,with expression being localized to certain tissues includingcardia/pylorus in the stomach, spleen peritrabeculae, osteoblasts, andhair follicles (“ECM” refers to extracellular matrix). Limitedexpression was also observed in tonsil and placenta (data not shown). Noexpression of huLRRC15 was observed in major organs (e.g., heart, liver,pancreas, lung).

Example 6. huLRRC15 is Expressed by Mesenchymal Stem Cells

Expression of huLRRC15 was measured by Western blot protein analysis ofcancer-associated fibroblast (CAF) cells that originate from a breastcancer patient, or commercial mesenchymal stem cell (MSC) lysates usingbiotinylated anti-huLRRC15 antibody muAD210.40.9 as shown in FIG. 9A.huLRRC15 was observed to be upregulated in a breast CAF lysate sampleupon treatment with TGFβ with low or negligible detectable expression inthe absence of TGFβ. By contrast, huLRRC15 expression was higher in theabsence of TGFβ in all three MSC lysate samples, and this expression wassignificantly upregulated upon TGFβ treatment. As previously discussed,MSCs are believed to make up a significant component of the cancerassociated fibroblast population in the tumor stroma (Cirri, P andChiarugi P. American Journal of Cancer Research 2011; 1 (4): 482-497).

Similar upregulation of huLRRC15 expression was observed by flowcytometry of two commercial mesenchymal stem cell populations upontreatment with TGFβ (FIGS. 9B, 9C). In human BM-MSC (Lonza), using CD29,CD44, CD105, CD166 as positive MSC markers and CD14, CD34, and CD45 asnegative MSC markers, a significant positive shift in the MSC populationexpressing huLRRC15 was observed upon treatment with TGFβ as comparedwith isotype (FIG. 9B). Correspondingly, in murine Balb/c BM-MSC(Cyagen), using CD29, CD44, CD34, and Sca-1 as positive MSC markers andCD117 as a negative MSC marker, a significant increase in the MSCpopulation expressing muLRRC15 was observed upon treatment with TGFβ ascompared with isotype (FIG. 9C).

Example 7. huLRRC15 is Associated with Cells UndergoingEpithelial-Mesenchymal Transition

The epithelial-mesenchymal transition (EMT) is a cellular mechanismwhich is believed to confer cellular plasticity on cancer cells. Thistransition to a more mesenchymal phenotype is thought to increase themotility and invasiveness of a primary cancer cell, potentially leadingto cancer metastatis, drug resistance, or evasion of the immune system.See, e.g., Ye, X. and Weinberg, R. A. Trends in Cell Biology, 2015, 25(11), pages 675-686. Data provided in this Example demonstrate thatcancer cells that had undergone EMT had an increased expression ofhuLRRC15 relative to their parental epithelial cancer cells.

FIG. 10 depicts the effect as determined by Western blot analysis oftreating baseline negative A549 (lung cancer) or PANC1 (pancreaticcancer) cells with TGFβ or StemXVivo™ EMT Inducing Media Supplement(“EMT Kit,” Catalog #CCM017, R&D Systems) to induce EMT. Proteinsrecognized as hallmarks of EMT, including N-cadherin, Snail, TCF8/ZEB1,increased expression, and proteins indicative of epithelial cellcharacteristics such as E-cadherin decreased expression. Expression ofthe housekeeping protein and protein loading control GAPDH did notchange significantly. huLRRC15 expression (measured using anti-LRRC15antibody muAD210.40.9) was observed to have increased in both A549 andPANC1 cells treated with either TGFβ or EMT Kit, and that havesubsequently undergone EMT.

FIGS. 11A-11C show that huLRRC15 expression increased in cells that haveundergone EMT, with the reverse mesenchymal-epithelial transition (MET)process occurring with the removal of EMT inducers. FIG. 11A shows A549or PANC1 cells untreated, or treated with TGFβ or EMT Kit for 5 days. InA549 or PANC1 cells, EpCAM expression, indicating epithelial-likecharacter, was highest in untreated cells. After treatment with TGFβ orEMT Kit, huLRRC15 (as measured with AF647-labeled huM25) in both celltypes was induced, while the epithelial marker EpCAM was reduced,suggesting a transition to a more mesenchymal phenotype (huLRRC15positive). FIG. 11B depicts the morphology of A549 cells treated withTGFβ (10 ng/mL continuously for 9 days) (top left), showing elongatedcells with fibrotic-like processes which appear mesenchymal-like, whileA549 cells treated with TGFβ (10 ng/mL continuously for 5 days) and thenwashed to remove the TGFβ no longer exhibited the mesenchymal cellmorphology after an additional 4 days (corresponding 9 days total)(bottom left). Hence, the EMT induced by TGFβ was reversible upon itsremoval. The induction of EMT by TGFβ and reversal of mesenchymal-likeproperties was also observed by flow cytometry (FIG. 11B). An increasein huLRRC15 expression (indicating more mesenchymal-likecharacteristics) (upper middle), and a decrease in EpCAM expression(indicating less epithelial-like characteristics) (upper right) wasobserved after treatment with TGFβ or EMT Kit. Upon discontinuation ofTGFβ or EMT Kit treatment, levels of huLRRC15 reverted to that observedat baseline (lower graphs).

FIG. 11C depicts the increase in mesenchymal character, andcorresponding decrease in epithelial character, of cells treated withTGFβ, as indicated by huLRRC15 (top graphs) and EpCAM levels (bottomgraphs) in A549 (left graphs) or PANC1 (right graphs) cells in vitro. Inboth A549 and PANC1 cells, huLRRC15 expression increased upon treatmentof cells with TGFβ or EMT Kit over 9 days, while EpCAM expression, anindicator of epithelial character, decreased. Consistent with the cellmorphology data in FIG. 11B, the protein expression of huLRRC15 andEpCAM indicated the huLRRC15-positive mesenchymal cells reverted to anepithelial-like state after removal of TGFβ or EMT Kit, and additionalcell culturing over 4 days.

Example 8. Preparation of Heterogeneous DAR huM25-vcMMAE ADCs

A huM25-val-cit-MMAE ADC composition heterogeneous in DAR was preparedby a two-step chemical process: disulfide reduction of huM25 followed byalkylation (conjugation) with maleimidocaproyl valine-citrulline(“val-cit”) para-aminobenzyl alcohol (“PABA”) monomethyl auristatin E(referred to herein as “vcMMAE”), illustrated below:

In the first step, a limited number of interchain disulfide bonds ofhuM25 are reduced with tris(2-carboxyethyl) phosphine (“TCEP”) (≥0.8equiv). Partially-reduced huM25 is then conjugated to vcMMAE (≥1.8equiv) in DMSO. Residual unreacted vcMMAE is quenched withN-acetyl-L-cysteine.

The top panel of FIG. 12 shows a chromatographic resolution of theresultant ADC preparation. As can be seen, the resultant ADC preparationis a heterogeneous mixture containing antibodies having zero MMAEmolecules attached (“DAR0” peak), two MMAE molecules attached (“DAR2”peak), four MMAE molecules attached (“DAR4” peak), six MMAE moleculesattached (“DAR6” peak) and eight MMAE molecules attached (“DAR8” peak)and has an average DAR of 4. Using huM25 as an example, specific ADCpreparations that comprise heterogeneous mixtures having an average DARof 4 are designated herein with “DAR4,” e.g., huM25-vcMMAE-DAR4.

Example 9. Preparation of huM25-vcMMAE ADCs Enriched in DAR2

Preparations of huM25-vcMMAE ADCs enriched DAR2 (referred to herein as“huM25-vcMMAE-E2”) were obtained via hydrophobic interactionchromatographic (“HIC”) resolution of the heterogeneous DAR ADCcomposition of Example 8. General methods for separating heterogeneousADC mixtures and isolating specific homogeneous species such as the DAR2and DAR4 peaks via HIC are described by Hamblen et al., 2004, ClinCancer Res 10:7063-7070.

A chromatogram of the enriched huM25-vcMMAE E2 ADC preparation is shownin the bottom panel of FIG. 12. The preparation is approximately 98%pure in the DAR2 ADC. Using huM25 as an example, specific ADCpreparations enriched in DAR2 are designated herein with “E2,” e.g.,huM25-vcMMAE-E2.

For the preparation of huM25-vcMMAE-E2, the heterogeneous DAR ADCmaterial as described in Example 8 was adjusted to column-binding saltconditions by the addition of ⅓ volume of 4.5M (NH₄)₂SO₄ to give 110 mSconductivity. This load material was pumped onto a 2.6×150-cm columnpacked with 70 mL GE Butyl Sepharose-HP resin and equilibrated withBuffer A [1.5M (NH₄)₂SO₄, 20 mM sodium phosphate, pH 7], using a GEÄKTAprime plus liquid chromatography system. After loading and washingto baseline, unconjugated antibody huM25 (“DAR0”) was eluted with a 90mS step-gradient blend of Buffers A and B (Buffer B=20 mM sodiumphosphate, pH 7+25% isopropanol) (retention time=3 min). Next,huM25-vcMMAE-E2 was prepared by elution with a 60 mS step-gradient blendof Buffers A and B (retention time=4 min). The eluted pool of materialenriched in huM25-vcMMAE-DAR2 was buffer exchanged and concentrated on aPellicon® tangential-flow filtration system (membrane XL-30 kD) using 15mM MES buffer pH 6.0 to afford the E2 preparation. Preparations of “E4”(enriched preparation of huM25-vcMMAE containing 4 MMAE molecules) and“E6” (enriched preparation of huM25-vcMMAE containing 6 MMAE molecules)and “E8” (enriched preparation of huM25-vcMMAE containing 8 MMAEmolecules) can also be isolated with this gradient. Final material wasquantified via absorbance at 280 nm, assessed for purity via HIC, andassessed for aggregation via size-exclusion chromatography (“SEC”).

Example 10. huM25-vcMMAE-E2 ADC has Potent Efficacy Against LRRC15Stromal(+)/Cancer(−) Tumors

The potent anti-tumor activity of an exemplary anti-huLRRC15 ADC,huM25-vcMMAE-E2, against huLRRC15 stromal(+)/cancer(−) tumors wasdemonstrated in a xenograft model with EBC-1 (human squamous NSCLC)cells. For the experiments, five million cells (EBC-1) that were grownin vitro were inoculated subcutaneously per mouse into the right flankof female SCID-Beige mice. Tumors were size matched at ˜200 mm³, anddosed intraperitoneally (IP) Q4D×6 (1 dose given every 4 days for atotal of 6 doses) as shown in FIG. 13A. Measurements of the length (L)and width (W) of the tumors were taken via electronic caliper and thevolume was calculated according to the following equation: V=L×W²/2. Inmice treated at 6 mg/kg, maximum tumor growth inhibition (TGI_(max)) of97% and tumor growth delay (TGD) of 305% was noted for huM25-vcMMAE-E2treatment, which was significantly improved (p<0.001) compared totreatment with isotype control antibody or corresponding isotype controlconjugate administered at the same regimen (FIG. 13A).

Other exemplary anti-huLRRC15 ADCs, huAD208.4.1-vcMMAE-DAR4,huAD208.14.1-vcMMAE-DAR4, and huM25-vcMMAE-DAR4 are also active in thisEBC-1 xenograft model. For this experiment, tumors were size matched at˜200 mm³, and dosed intraperitoneally (IP) Q7D×2 (1 dose given every 7days for a total of 2 doses) as shown in FIG. 13C. In mice treated at 3mg/kg, huAD208.4.1-vcMMAE-DAR4, huAD208.14.1-vcMMAE-DAR4 andhuM25-vcMMAE-DAR4 exhibited TGI_(max) values of 90%, 84% and 88%,respectively. Tumor growth delays of 129%, 114% and 129% was noted forhuAD208.4.1-vcMMAE-DAR4, huAD208.14.1-vcMMAE-DAR4 and huM25-vcMMAE-DAR4respectively, which was significantly improved (p<0.001) compared totreatment with isotype control antibody or corresponding isotype controlconjugate administered at the same regimen (FIG. 13C).

The potent anti-tumor activity of huM25-vcMMAE-E2 was also demonstratedin a xenograft model with HPAF-II (human pancreatic) cells. For theexperiment, 1 million cells (HPAF-II) that were grown in vitro wereinoculated subcutaneously per mouse into the right flank of femaleSCID-Beige mice. Tumors were size matched at ˜200 mm³, and dosedintraperitoneally (IP) Q4D×6 (1 dose given every 4 days for a total of 6doses) as shown in FIG. 13B. In mice treated at 6 mg/kg, a TGI_(max) of76% and a tumor growth delay of 133% was noted, which was significantlyimproved (p<0.001) compared to treatment with isotype control antibodyor corresponding isotype control conjugate administered at the sameregimen (FIG. 13B).

The anti-huLRRC15 ADC huM25-vcMMAE-E2 displays potent anti-tumoractivity in differing tumor models (FIG. 13A, 13B). Anti tumor efficacywas observed for several anti-huLRRC15 ADCs (FIG. 13C). The efficaciesof huAD208.4.1-vcMMAE-DAR4, huAD208.14.1-vcMMAE-DAR4 andhuM25-vcMMAE-DAR4 were similar in magnitude (TGI_(max)) and duration(TGD) when dosed at 3 mg/kg in EBC1 tumors (FIG. 13C). The efficacieswere significantly improved (p<0.001) compared to treatment with anisotype control antibody or a corresponding isotype control conjugateadministered at the same regimen.

No anti-tumor activity was observed with huM25-vcMMAE-DAR4 in thexenograft model HCC-827-ER (NSCLC), even though robust huLRRC15expression was observed by IHC (3+ score). In this experiment, 2 millioncells (HCC-827-ER) that were grown in vitro were inoculatedsubcutaneously per mouse into the right flank of female SCID-Beige mice.Tumors were size matched at ˜200 mm³, and dosed intraperitoneally (IP)Q4D×6 (1 dose given every 4 days for a total of 6 doses), and nosignificant anti-tumor activity was observed (FIG. 13D).

Example 11. Anti-huLRRC15 ADCs are Active Against Large Tumors thatRegrow Following Earlier Rounds of Treatment

To demonstrate that tumors that regrow post treatment with anti-huLRRC15ADCs are sensitive to anti-huLRRC15 ADCs, tumors were treated with ananti-huLRRC15 ADC, permitted to regrow and retreated. For theexperiment, SUM190PT human breast cancer cells (developed from a primaryhuman ER negative and PR negative breast cancer tumor) were grown topassage three in vitro. Five million cells per mouse were inoculatedsubcutaneously into the right flank of female SCID mice. Tumors weresize matched at ˜250 mm³, antibodies and immunoconjugates wereadministered IP Q4D×4 (one dose give every 4 days for a total of 4doses) at 10 or 3 mg/kg, respectively. At Day 70 post sizematch whentumors had regrown to ˜650 mm³, animals were retreated withhuM25-vcMMAE-DAR4 IP Q4D×6 (one dose given every 4 days for a total of 6doses) at 6 mg/kg. Some large tumors at time of retreatment wereharvested for immunohistochemistry assessment of huLRRC15 expression.

Results are shown in FIG. 14. Maximum tumor growth inhibition(TGI_(max)) of 77% was observed for initial treatment withhuM25-vcMMAE-DAR4, which was significantly (p<0.001) better than theisotype control antibody or corresponding isotype control conjugateadministered at the same regimen.

Following retreatment after growth, tumor regression was again observed,indicating tumors remained sensitive to huM25-vcMMAE-DAR4, with anoverall TGD of 133% (p<0.01). Expression of huLRRC15 was retained inthese previously treated tumors. These findings suggest that becausehuLRRC15 is expressed on non-cancerous fibroblasts within the tumor, thehuLRRC15 antigen is not under the same genetic selective pressure as thecancer cells, allowing the tumors to retain sensitivity to anti-huLRRC15ADCs.

Example 12. Anti-huLRRC15 ADCs Exert Anti-Cancer Activity AgainsthuLRRC15 Stromal(+)/Cancer(−) Tumors at Least in Part Via BystanderEffect

The fact that anti-huLRRC15 ADCs comprising cell permeable cytostaticand/or cytotoxic agents effect anti-tumor activity via a bystanderkilling effect was demonstrated in EBC-1 squamous lung cancer xenograftswith two anti-huLRRC15 ADCs: huM25-vcMMAE-E2 and huM25-mcMMAF-E2. MMAEis capable of traversing cell membranes. Closely related monomethylauristatin F (“MMAF”) is not.

The ADC huM25-mcMMAF-E2 was prepared in a manner analogous to theprotocol described for huM25-vcMMAE-E2 in Examples 8 and 9. Thestructure of the linker-drug moiety mcMMAF is:

For the experiments, 5 million EBC-1 cells grown in vitro were implantedper mouse into the right flank of SCID-beige mice. Tumors weresize-matched at ˜300 mm³ and treated on day 0 and day 4 with 6 mg/kgintraperitoneally for each biologic drug shown in FIG. 15A. Tumors wereharvested from the mice in each group 11 days post randomization andstart of treatment. Tumors were dissociated into single cell suspensionsusing Miltenyi Biotec gentleMACS Dissociator according to themanufacturer's guidelines for the dissociation of tough tumors.Dissociated tumor cells were passed through a 70 μm filter, counted, andimmediately used for flow cytometry. Single-cell suspensions wereresuspended in PBS with 10% FBS and 10 μg/mL FcR block (anti-mouseCD16/CD32, clone 93, eBioscience, Cat.#16-0161.) The following directlyconjugated antibodies were used for flow cytometry: human IgG₁ κ isotypecontrol (AB095) AF647, anti-huLRRC15 (hu208.4.1a. 1a) AF647, anti-FAP(huFAP MAB5) AF647, mouse anti-human CD326 (EPCAM) PE (clone 1B7,eBioscience, Cat.#12-9326), anti-mouse CD11b PE (clone M1/70,eBioscience, Cat.#12-0112), anti mouse CD11c PE (clone N418,eBioscience, Cat.#12-0114), and anti-mouse F4/80 PE (clone BM9,eBioscience, Cat.#12-4801). Ex vivo EBC1 tumor cell suspensions wereincubated with fluor-conjugated antibodies for 20 minutes on ice andwashed twice using PBS with 1% FBS. Flow cytometry data were collectedusing a Becton Dickinson FacsCalibur, and data were analyzed usingFlowJo™ analysis software (TreeStar). The ex vivo flow cytometry data(FIGS. 15B-15E) are shown as a percentage of cells gated positive foreach antigen relative to total live cells gated via FSC/SCC. It shouldbe noted that percentages reported are relative values to the total exvivo EBC1 tumor and that a reduction in one cell population (e.g., EPCAMpositive cancer cells) would result in an inverse increase in remainingcell populations when this analysis method is used.

The data shown in FIGS. 15B-15E demonstrate that huM25-vcMMAE-E2 canresult in cancer cell killing of cancer cells (e.g., EBC-1) which do notexpress the huLRRC15 antigen by targeting the delivery of the ADC to theneighboring huLRRC15 stromal positive cancer associated fibroblasts.When tumors were treated with huLRRC15 targeted ADCs containing thenon-cell permeable highly structurally related payload MMAF, there wasno in vivo anti-tumor activity. The cancer associated fibroblastpopulation which is known to be huLRRC15 positive (FIG. 7), does notshow a decrease in the percentage of fibroblasts found within the tumor(FIG. 15C). Therefore, the fibroblasts are acting as targeting andprocessing cells for the MMAE payload, which has a more profound growthinhibitory effect on the cancer cells than on the huLRRC15-expressingstromal fibroblasts. An increase in immune infiltrate (CD11c or F4/80positive cells) was noted post treatment (FIGS. 15D-15E), indicatingthat this ADC increases the expression of specific immune cellpopulations within the tumor (e.g., antigen presenting cells). Thefinding that in vivo anti-huLRRC15 ADCs (e.g., huM25-vcMMAE-E2) increasethe expression of specific immune populations within the tumor provideclear rationale for combining anti-huLRRC15 ADCs with immune targetedtherapies.

Dissociated single-cell suspensions of the ex vivo EBC1 tumors shown inFIG. 15A were seeded at 120,000 live cells into tissue culturemicroscopy chamber slides. Cells were allowed to sit and incubate for 48hours, and then fixed in 2% paraformaldehyde for 15 min at 37° C. Thecells were washed with PBS and permeabilized using 0.3% Triton-X in PBSat 37° C. for 30 min. Cells were washed with PBS and blocked in 5% BSAfor 30 min at 37° C. Cells were incubated with anti-α-SMA (clone 1A4DAKO, Cat.#36962) and then detected with AlexaFluor AF594 (LifeTechnologies, Cat.# A21203) secondary antibody (2 μg/mL) for 30 min at37° C. Subsequently, cells were stained using anti-human EpCAM-AF488(Cell Signaling Technology, Cat.#5198) and washed 3× in PBS. Finally theslides were removed from the chambers and mounted with a coverslip usingProLong Diamond Anti-Fade with DAPI (Life Technologies, Cat.#36962).Image acquisition was performed using Zeiss Axiovision Software on aZeiss Axiovert 200M inverted fluorescent microscope. Statisticaldifferences between groups were determined using unpaired T tests inGraphPad Prism™. Representative images and graphing of cancer cell andfibroblast populations as counted by microscopy are shown in FIG. 15F.Similar to the observations in FIGS. 15B-15E, a significant decrease incancer cells was noted following the treatment of EBC-1 tumors withhuM25-vcMMAE-E2, while a fibroblast population continued to persist.Further, phosphohistone H3 (pHH3) staining indicated that after 72hours, a higher number of mitotic figures were evident afterhuM25-vcMMAE-E2 treatment (FIG. 15G), indicating cell cycle arrest inmitosis. The percentage of pHH3 positive cells decreased with additionaldosing and longer time (FIG. 15H) correlating with cancer cell death andtumor shrinkage (FIG. 15A). EBC-1 tumors at day 11 post-treatment showedenhanced staining for CD45 and F4/80 after treatment withhuM25-vcMMAE-E2 as compared with isotype ADC (FIG. 15I).

The data shown in FIG. 15A-15I suggest that huM25-vcMMAE-E2 works atleast in part via huLRRC15 targeted bystander activity from the MMAEpayload. MMAE is a cell permeable payload which allows it to pass acrosscell membranes without active transport. The data shown suggest thatMMAE ADCs that bind to huLRRC15 are internalized by the stromalfibroblasts releasing free MMAE which can then be passively taken up byneighboring cancer cells in a bystander fashion resulting in potentcancer cell killing. Localizing anti-huLRRC15 ADCs containing MMAE at ahigher concentration within cancer stroma may also result in somenon-specific release and cleavage of the valine-citrulline linkerextracellularly, which can then cross cancer cell membranes and causecell death. When the non-cell permeable payload MMAF was conjugated tohuM25 and tested in EBC-1 cells, there was no apparent activity,suggesting the cell permeable properties of MMAE is required forhuM25-vcMMAE-E2's targeted bystander in vivo activity.

A panel of human tumors shown in FIG. 16 was stained by IHC for theproliferation marker Ki67. The Ki67 positive cancer and stromal cellpopulations within each tumor were separately counted and graphed. Thedata show that, in general, cancer cells within several distinct tumortypes (e.g., colon, breast, lung, pancreatic, ovarian, melanoma, renal)have a much higher proliferative rate (Ki67 positive) than stromalcells. Since the MMAE payload requires cells to enter mitosis in orderto induce mitotic arrest and subsequent cell death, the MMAE payload isgoing to be more efficacious against rapidly dividing cells (e.g.,cancer cells) than slower dividing stromal cells (e.g., fibroblasts).

The data shown in FIG. 17A demonstrate that the non-cell permeablepayload MMAF can kill huLRRC15 expressing cancer cells in vitro (e.g.,HCT116-huLRRC15) when conjugated to huM25 (EC₅₀=0.028 nM), with apotency that is similar to that seen for huM25-vcMMAE-E2 (EC₅₀=0.069nM). These data therefore demonstrate that the huM25-mcMMAF ADC iscapable of being efficiently internalized and processed within a cell torelease the highly potent anti-mitotic payload MMAF. However, when thissame huM25-mcMMAF-E2 ADC was tested in vivo in the PANC1 (3+huLRRC15stromal positive, cancer negative) tumor model (FIG. 17B), the ADC didnot display any in vivo activity, while huM25-vcMMAE-E2 exhibited potentanti-tumor efficacy (TGI_(max) of 94.4%). These findings suggest thathuM25-vcMMAE-E2 works at least in part via huLRRC15 targeted bystanderactivity that relies on MMAE's cell permeable characteristics to killthe cancer cells within the tumor mass.

Example 13. E2 ADCs have Equal or Better Therapeutic Index

The safety profiles of E2, E4 and E2/E4 preparations of huM25-vcMMAEADCs were assessed in a rat tolerability experiment (FIG. 18A-18B). Forthe assay, Sprague Dawley wild type rats were dosed with a single IVdose at MMAE equivalent levels of each antibody drug conjugate. Deathoccurred earlier (day 3/4) and at a higher percentage (50%) withhuM25-vcMMAE-E4 than was seen for huM25-vcMMAE-E2 (day 8, 25%). Doublethe protein antibody dose was delivered with equal amounts of MMAE, andimproved survival for E2 was seen over E4. Fewer deaths occurred in theanimals dosed with E2 than E4 ADCs, and the deaths that did occurhappened later on Day 8. Broad drug distribution DAR4 has an MTD of 20mg/kg. Weight loss was not significant for huM25-vcMMAE-E2 when dosed at60 mg/kg, but there was increased weight loss for rats dosed at the MMAEequivalent dose level of 30 mg/kg with huM25-vcMMAE-E4 (FIG. 18B). Thisobservation demonstrates that higher drug loaded anti-huLRRC15 ADCs arenot as well tolerated as when a higher protein dose is delivered with alower MMAE drug antibody ratio such as 2 cytostatic and/or cytotoxicagents per antibody (E2).

Four preparations of huM25-vcMMAE that differed in their relative DARprofile (E2, E2/E4, E4 or DAR4) were tested for efficacy in EBC1 tumors.5 million cells were implanted subcutaneously into SCID/Beige mice, andmice were randomized when tumor group mean volumes reached ˜200 mm³.HuM25-vcMMAE was administered intraperitoneally at MMAE-equivalent dosesevery seven days for a total of two doses. Maximum tumor growthinhibition (TGI_(max)) of ≥89% and tumor growth delay of 123% was notedin all groups treated with huM25-vcMMAE, which was significantly(p<0.001) better than the isotype control antibody or correspondingisotype control conjugate administered at the same regimen (FIG. 19).All doses were well tolerated and no significant body weight reductionswere observed (data not shown). Efficacy in the EBC1 tumor model forhuM25-vcMMAE-E2 was comparable to huM25-vcMMAE-E2E4 (i.e., the E2/E4preparation of huM25-vcMMAE), huM25-vcMMAE-E4 and huM25-vcMMAE-DAR4indicating that higher order DAR is not required for optimal anti-tumorpotency and that higher antibody dosing is possible withhuM25-vcMMAE-E2. huM25-vcMMAE-E2 had comparable or slightly betterefficacy than huM25-vcMMAE-DAR4 in similar xenograft assays carried outwith NCI-H226, PANC1 and HPAF-II tumors (data not shown).

The ability to administer more MMAE by dosing a higher total antibodydose with a lower drug antibody ratio (DAR) of E2 resulted in lesstoxicity than when dosing with a lower antibody dose with a higher drugantibody ratio (e.g., E4) was shown in rat tolerability studies (FIG.18A, 18B). Also FIG. 19 shows that using a lower drug-antibody ratio ofE2 provides just as much anti-tumor efficacy as ADC preparationscontaining a higher drug load (e.g., E2/E4, DAR4, E4) when dosed at MMAEequivalent levels. Taken together these data demonstrate that animproved therapeutic index can be achieved by using anti-huLRRC15 ADCscontaining 2 drug/linkers per antibody compared to an ADC preparationcontaining a higher DAR (e.g., DAR4, E4). These data suggest thatanti-huLRRC15 ADCs containing a lower DAR will be able to be dosedclinically at higher levels than ADCs containing a higher DAR (e.g.,DAR4 or E4) and will have an improved therapeutic index.

Example 14. Anti-huLRRC15 ADCs are Superior to Current Standards of Care

The potency of anti-huLRRC15 ADCs as compared to current standards ofcare was assessed in xenograft models with NCI-H1650 (squamous NSCLC),HN5 (head and neck), EBC-1 (squamous NSCLC), NW231 (breast) and PANC1(pancreatic) cancer cells. Anti-huLRRC15 ADC huM25-vcMMAE-E2 wascompared to erlotinib, carboplatin, cetuximab, doxorubicin andgemcitabine. Standard of care agents were dosed at maximally efficaciousor maximally tolerated dose levels. For FIG. 20A, NCI-H1650 cells (5million cells) were implanted subcutaneously into SCID/Beige mice andtumors were randomized when they reached ˜200 mm³ and dosed withbiologics at 6 or 12 mg/kg intraperitoneally on a Q4D×6 (one dose givenevery 4 days for a total of 6 doses), while erlotinib was dosed orallydaily for 10 days at 100 mg/kg, and carboplatin was dosedintraperitoneally Q4D×4 (one dose given every 4 days for a total of 4doses) at 50 mg/kg. The anti-tumor efficacy for huM25-vcMMAE-E2(TGI_(max) of 92%, TGD of 450%) was superior to that seen for eithererlotinib (TGI_(max) of 90%, TGD of 77%) or carboplatin (TGI_(max) of56%, TGD of 77%).

In FIG. 20B, HN5 cells (5 million) were implanted subcutaneously intoNSG mice, and mice were randomized when the tumor group mean volumesreached ˜200 mm³ and dosed with isotype mAb or ADCs at 4.5 mg/kgintraperitoneally on a Q4D×6 (one dose given every 4 days for a total of6 doses), while erlotinib was dosed orally daily for 10 days at 150mg/kg, and cetuximab was dosed intraperitoneally Q7D×3 (one dose givenevery 7 days for a total of 3 doses) at 3 mg/kg. The anti-tumor efficacyfor huM25-vcMMAE-E2 (TGI_(max) of 96%, TGD of 194%) was superior to thatseen for either erlotinib (TGI_(max) of 23%, TGD of 12%) or cetuximab(TGI_(max) of 73%, TGD of 59%).

In FIG. 20C, EBC1 cells (5 million) were implanted subcutaneously intoSCID/Beige mice and mice were randomized when the tumors reached ˜200mm³ and dosed with biologics at 6 mg/kg intraperitoneally on a Q7D×2(one dose given every 7 days for a total of 2 doses), while erlotinibwas dosed orally daily for 10 days at 50 mg/kg, and carboplatin wasdosed intraperitoneally Q4D×3 (one dose given every 4 days for a totalof 3 doses) at 50 mg/kg. The anti-tumor efficacy for huM25-vcMMAE-E2(TGI_(max) of 92%+, TGD of 129%) was superior to that seen for eithererlotinib (TGI_(max) of 27%, TGD of 14%) or carboplatin (TGI_(max) of39%, TGD of 43%).

In FIG. 20D, NW231 breast cancer cells (10 million) were implantedsubcutaneously into SCID mice, and mice were randomized when the tumorsreached ˜150 mm³ and dosed with biologics at 12 mg/kg intraperitoneallyon a Q4D×6 (one dose given every 4 days for a total of 6 doses), whiledoxorubicin was dosed intravenously at 1 mg/kg on days 0, 4, 8, 13 and18. The anti-tumor efficacy for huM25-vcMMAE-E2 (TGI_(max) of 89%) wassuperior to that seen for doxorubicin (TGI_(max) of 65%).

In FIG. 20E, PANC1 pancreatic cancer cells (10 million) were implantedsubcutaneously into SCID mice, and mice were randomized when the tumorsreached ˜125 mm³ and dosed with biologics at 12 mg/kg intraperitoneallyon a Q4D×6 (one dose given every 4 days for a total of 6 doses), whilegemcitabine was dosed intraperitoneally at 100 mg/kg Q3D×4) (one dosegiven every three days for 4 doses total). The anti-tumor efficacy forhuM25-vcMMAE-E2 (TGI_(max) of 94%) was superior to that seen forgemcitabine (TGI_(max) of 55%).

The in vivo efficacy data shown in FIG. 20A-20E provides examples ofwhere anti-huLRRC15 ADCs such as huM25-vcMMAE-E2 outperformed standardof care agents (e.g., carboplatin, erlotinib, gemcitabine, cetuximab,doxorubicin) commonly used in cancer therapy. This data suggests thatanti-huLRRC15 ADCs (e.g., huM25-vcMMAE-E2) may be more clinicallyefficacious than certain commonly used anti-cancer therapies.

Example 15. Anti-huLRRC15 ADCs are Active when Administered Adjunctiveto Cytotoxic Anti-Cancer Treatments

The anti-tumor efficacy of anti-huLRRC15 ADCs administered adjunctive toradiation and other non-targeted chemotherapeutic agents (e.g.,gemcitabine, docetaxel, carboplatin) was demonstrated in xenograftmodels with HPAF-II (pancreatic), EBC-1 (squamous NSCLC) and SCC-15(head and neck) cells. Improved antitumor activity was seen whenanti-huLRRC15 ADCs (e.g., huM25-vcMMAE-E2) were adjunctivelyadministered with these cytotoxic anti-cancer treatments. The efficacyof adjunctive treatment was better than the efficacy seen for eitherdrug alone.

In FIG. 21A, HPAF-II pancreatic cancer cells (1 million) were implantedsubcutaneously into SCID mice, and mice were randomized when the tumorsreached ˜200 mm³ and dosed with biologics at 6 mg/kg intraperitoneallyon a Q4D×6 (one dose given every 4 days for a total of 6 doses), whilegemcitabine was dosed intraperitoneally at 100 mg/kg (Q3D×4)×2 (one dosegiven every three days for 4 doses per cycle, 2 cycles with 8 totaldoses given). In this model at the doses tested, the anti-tumor efficacyseen for huM25-vcMMAE-E2 (TGI_(max) of 57%, TGD of 57%) and gemcitabine(TGI_(max) of 73%, TGD of 117%) increased when used adjunctively(TGI_(max) of 85%, TGD of 183%) to achieve better efficacy than was seenfor either single agent alone.

In FIG. 21B, EBC-1 squamous NSCLC cells (5 million) were implantedsubcutaneously into SCID/Beige mice, and mice were randomized when thetumors reached ˜200 mm³ and dosed with biologics at 6 mg/kgintraperitoneally on a Q7D×2 (one dose given every 7 days for a total of2 doses), while gemcitabine was dosed intraperitoneally at 100 mg/kg(Q3D×3) (one dose given every three days for 3 doses total). In thismodel at the doses tested, the anti-tumor efficacy seen forhuM25-vcMMAE-E2 at 3 mg/kg (TGI_(max) of 91%, TGD of 144%) and docetaxel(TGI_(max) of 77%, TGD of 48%) increased when used adjunctively(TGI_(max) of 98%, TGD of 348%) to achieve better efficacy than was seenfor either single agent alone.

The results shown in FIG. 21C highlight the improved activity whenanti-huLRRC15 ADCs (e.g., huM25-vcMMAE-DAR4) are administered adjunctivewith docetaxel. EBC-1 squamous NSCLC cells (5 million) were implantedsubcutaneously into SCID/Beige mice, and mice were randomized when thetumors reached ˜200 mm³ and dosed with biologics at 3 or 6 mg/kgintraperitoneally on a Q4D×6 (one dose given every 4 days for a total of6 doses), while docetaxel was dosed intravenously once at 7.5 mg/kgQ3D×3 (one dose given every 3 days for a total of 3 doses). In thismodel at the doses tested, the anti-tumor efficacy seen forhuM25-vcMMAE-DAR4 (TGI_(max) of 92%, TGD of 129%) and gemcitabine(TGI_(max) of 95%, TGD of 171%) increased when used adjunctively(TGI_(max) of 98%, TGD of 533%) to achieve better efficacy than was seenfor either single agent alone.

Radiation is a commonly used cytotoxic therapy in oncology. Data shownin FIG. 21D highlight the improved activity when anti-huLRRC15 ADCs(e.g., huM25-vcMMAE-E2) are administered adjunctive to radiationtherapy. SCC-15 head and neck cancer cells (1 million) were implantedsubcutaneously into SCID mice, and mice were randomized when the tumorsreached ˜200 mm³ and dosed with biologics at 12 mg/kg intraperitoneallyon a Q4D×6 (one dose given every 4 days for a total of 6 doses), whilethe radiation was directed at the tumor with a single radiation dose of15 Gy on day 0. In this model at the doses tested, the anti-tumorefficacy seen for huM25-vcMMAE-E2 (TGI_(max) of 58%, TGD of 44%) andradiation (TGI_(max) of 66%, TGD of 88%) increased when usedadjunctively (TGI_(max) of 90%, TGD of 219%) to achieve better efficacythan was seen for either single agent alone.

The results shown in FIG. 21E highlight the improved activity whenanti-huLRRC15 ADCs (e.g., huM25-vcMMAE-E2) are administered adjunctiveto carboplatin. SCC-15 head and neck cancer cells (1 million) wereimplanted subcutaneously into SCID mice, and mice were randomized whenthe tumors reached ˜200 mm³ and dosed with biologics at 12 mg/kgintraperitoneally on a Q7D×6 (one dose given every 7 days for a total of6 doses), while carboplatin was dosed intraperitoneally once at 50 mg/kgQ4D×4 (one dose given every 4 days for a total of 4 doses). In thismodel at the doses tested, the anti-tumor efficacy seen forhuM25-vcMMAE-E2 (TGI_(max) of 58%, TGD of 44%) and carboplatin(TGI_(max) of 56%, TGD of 33%) increased when used adjunctively(TGI_(max) of 86%, TGD of 83%) to achieve better efficacy than was seenfor either single agent alone.

The data outlined in FIG. 21A-21E demonstrate that anti-huLRRC15 ADCsused adjunctive to cytotoxic anti-cancer agents (e.g., gemcitabine,docetaxel, carboplatin, radiation) have improved efficacy as compared toeach agent alone. This suggests that anti-huLRRC15 ADCs such ashuM25-vcMMAE-E2 when administered adjunctive to cytotoxic anti-canceragents may be more effective in cancer patients than either agent alone.

Example 16. Anti-huLRRC15 ADCs are Active when Administered Adjunctiveto Other Targeted Anti-Cancer Agents

The anti-tumor efficacy of anti-huLRRC15 ADCs administered adjunctive toother targeted chemotherapeutic agents (e.g., erlotinib, cetuximab,anti-PD-1 antibody) was demonstrated in xenograft models with NCI-H1650(adeno NSCLC), HN5 (head & neck), SCC-15 (head & neck) and MC38 (mousesyngeneic colorectal) cancer cells. Improved antitumor activity was seenwhen anti-huLRRC15 ADCs (e.g., huM25-vcMMAE-E2) were administeredadjunctive to these targeted anti-cancer treatments. The adjunctiveefficacy was better than the efficacy of either drug alone.

The results shown in FIG. 22A highlight the improved activity whenanti-huLRRC15 ADCs (e.g., huM25-vcMMAE-E2) are administered adjunctiveto erlotinib. NCI-H1650 NSCLC cancer cells (5 million) were implantedsubcutaneously into SCID/Beige mice, and mice were randomized when thetumors reached ˜200 mm³ and dosed with biologics at 6 or 12 mg/kgintraperitoneally on a Q4D×6 (one dose given every 4 days for a total of6 doses), while erlotinib was dosed orally daily at 100 mg/kg for 10doses. In this model at the doses tested, the anti-tumor efficacy seenfor huM25-vcMMAE-E2 (TGI_(max) of 92%, TGD of 450%) and erlotinib(TGI_(max) of 90%, TGD of 77%) increased when used adjunctively(TGI_(max) of 92%, TGD of >538%) to achieve better efficacy than wasseen for either single agent alone.

The results shown in FIG. 22B highlight the improved activity whenanti-huLRRC15 ADCs (e.g., huM25-vcMMAE-E2) are administered adjunctiveto cetuximab. SCC-15 head and neck cancer cells (1 million) wereimplanted subcutaneously into SCID mice, and mice were randomized whenthe tumors reached ˜200 mm³ and dosed with isotype antibodies or ADCs at12 mg/kg intraperitoneally on a Q7D×6 (one dose given every 7 days for atotal of 6 doses), while cetuximab was dosed intraperitoneally at 3mg/kg Q7D×3 (one dose given every 7 days for a total of 3 doses). Inthis model at the doses tested, the anti-tumor efficacy seen forhuM25-vcMMAE-E2 (TGI_(max) of 58%, TGD of 44%) and cetuximab (TGI_(max)of 66%, TGD of 58%) increased when used adjunctively (TGI_(max) of 87%,TGD of 125%) to achieve better efficacy than was seen for either singleagent alone.

The results shown in FIG. 22C highlight the improved activity whenanti-huLRRC15 ADCs (e.g., huM25-vcMMAE-E2) are administered adjunctiveto anti-PD-1 targeted agents, such as an anti-PD-1 antibody. Exemplaryanti-PD-1 antibodies include those described in U.S. provisionalapplication No. 62/394,314, such as the anti-PD-1 antibody having aheavy chain amino acid sequence of SEQ ID NO:91 or 92, and a light chainamino acid sequence of SEQ ID NO:93, used in this Example.

MC-38 mouse colorectal cancer cells (250,000) were implantedsubcutaneously into C57BL/6 mice, and mice were randomized when thetumors reached ˜100 mm³ and dosed with isotype antibodies or ADCs at 12mg/kg intraperitoneally on a Q4D×6 (one dose given every 4 days for atotal of 6 doses), while the anti-PD-1 antibody (“Anti-PD1 mAb”) wasdosed intraperitoneally at 2 mg/kg Q4D×6 (one dose given every 4 daysfor a total of 6 doses). In this model huM25-vcMMAE-E2 did not showsingle agent activity, while the anti-PD-1 antibody alone did displayefficacy (TGI_(max) of 67%). huM25-vcMMAE-E2 administered adjunctivelywith anti-PD-1 antibody was more efficacious (TGI_(max) of 87%) thaneither agent alone. Given that huM25-vcMMAE-E2 did not have single agentactivity in this model the improved activity when used adjunctively withanti-PD-1 antibody was unexpected. This novel finding suggests thatanti-huLRRC15 ADCs, when used adjunctively with immune modulating agentssuch as anti-PD-1 antibody may have improved anti-cancer activity overeither agent alone.

The data outlined in FIG. 22A-22C demonstrate that anti-huLRRC15 ADCsadministered adjunctive to targeted anti-cancer agents (e.g., anti-PD-1antibody, erlotinib, cetuximab) have improved efficacy as compared toeach agent alone. This suggests that anti-huLRRC15 ADCs such ashuM25-vcMMAE-E2 may be more effective clinically when used adjunctivelyto targeted anti-cancer agents in cancer patients than either agentalone.

Example 17. Anti-huLRRC15 ADCs Comprising DNA-Damaging Payloads areActive

The potent anti-tumor activity of anti-huLRRC15 ADCs comprisingDNA-damaging cytostatic and/or cytotoxic agents against several types ofhuLRRC15 stromal(+)/cancer(−) tumors was demonstrated with differentexemplary ADCs in xenograft models with EBC-1 (squamous NSCLC),NCI-H1650 (adeno NSCLC), HN-5 (head and neck), HPAF-II (pancreatic) andPANC-1 (pancreatic) cancer cells.

For the experiments, anti-huLRRC15 ADCs comprising apyrrolobenzodiazepine dimer (“PBD”) cytostatic and/or cytotoxic agentwere prepared by conjugating the PBD synthon illustrated below (“vaPBD”)with anti-huLRRC15 antibody huAD208.4.1 or an isotype control antibodyto yield a DAR of 2. Preparation of the huAD208.4.1-PBD-DAR2 ADC wasaccomplished according to the procedure described in Example 8.

In an analogous manner, PBD-containing ADCs were prepared with huM25comprising an engineered S239C mutation in the constant region(“huM25-S239C”) to allow for preferential generation of a DAR2 ADC withvaPBD shown above. As used herein, huM25-S239C refers to ananti-huLRRC15 antibody or ADC having a heavy chain amino acid sequenceaccording to SEQ ID NO:100 or 103, and a light chain amino acid sequenceaccording to SEQ ID NO:19. Accordingly, ADCs comprising the S239Cmutation did not require chromatographic separation in order to exhibitenrichment in ADCs having DAR2. Hence, the ADCs prepared comprising ahuLRRC15 antibody and S239C mutation, including those comprisinghuM25-S239C, are referred to herein as “E2.”

FIG. 23A shows that in vitro cell killing of 3T12-huLRRC15 transfectedcells by huLRRC15-targeting ADC huM25-S239C-PBD-E2 (i.e., the ADC formedwith huM25 variable domains with S239C variant in the Fc region to allowfor selective formation of DAR2 preparation of the vaPBD adduct) issignificantly higher than that of isotype ADC isotype-S239C-PBD-E2having the same Fc region and PBD drug displayed in the same manner.Such a result is presumably owing to the localization of the ADChuM25-S239C-PBD-E2 to the huLRRC15-expressing cell surface before beinginternalized and enzymatically processed to release the cytotoxic PBDagent.

Example 18. Anti-huLRRC15 PBD ADCs Kill Cells with MesenchymalProperties

As described above in Example 7, cancer cells that have undergone EMThad an increased expression of huLRRC15 as compared to cells that hadnot undergone EMT. Data provided in the present Example show that thisincreased level of huLRRC15 expression correlated with an increasedsensitivity to ADCs that targeted huLRRC15. As shown in FIGS. 23B and23C, anti-LRRC15 ADC huAD208.4.1-PBD-DAR2, which was cross-reactive tohuman and mouse LRRC15, demonstrated cell killing effects in vitroagainst mesenchymal stem cells expressing LRRC15. In human BM-MSC(Lonza) treated with 10 ng/mL TGFβ, huAD208.4.1-PBD-DAR2 exhibited ahigher cell killing effect than isotype-PBD-DAR2 at the same doses (FIG.23B). A similar in vitro cell killing effect profile was also observedin murine Balb/c BM-MSC (Cyagen) treated with TGFβ (10 ng/mL) and eachof the ADCs (FIG. 23C).

Additionally, FIG. 23D depicts experiments in which anti-huLRRC ADCskilled A549 lung cancer cells that have undergone epithelial-mesenchymaltransition (EMT). Standard A549 cells did not show a viabilitydifference after treatment with isotype-S239C-PBD-E2 orhuM25-S239C-PBD-E2 (top graph). However, in A549-EMT transformed cellstreated with TGFβ, the huLRRC15-specific ADC huM25-S239C-PBD-E2exhibited a significantly higher cell killing effect than isotype ADC(EC₅₀=0.01 nM vs. 2.3 nM for isotype ADC) (bottom graph).

Example 19. Anti-huLRRC15 PBD ADCs Exhibit In Vivo Anti-Tumor Effects

The above Examples show that huLRRC15 expression was increased incertain cancer cells that have undergone EMT, and those cells were moresensitive to huLRRC15 ADCs in vitro. The data provided in the presentExample demonstrate that the same huLRRC15 ADCs exhibit significant invivo efficacy. FIG. 24A depicts the effect of treating SCID mice withEBC-1 squamous NSCLC tumors. EBC-1 squamous NSCLC cells (5 million) wereimplanted subcutaneously into SCID mice, and mice were randomized whenthe tumors reached ˜175 mm³ and dosed with ADC or isotype antibody at0.6 mg/kg intraperitoneally on day 0. With a single dose of ADC,huM25-S239C-PBD-E2 demonstrated significant anti-tumor effect after 13days as compared to the same dose of isotype-S239C-PBD-E2 (p<0.01).

FIGS. 24B-24D depicts immunohistochemistry results from the in vivoexperiment depicted in FIG. 24A, consistent with an immunologicalanti-tumor response. FIG. 24B shows images of exemplary tumor slicesstained for α-SMA, a cancer-associated fibroblast marker, at 1× (toppictures) and 20× (bottom pictures) magnification. The samples weretaken from mice dosed with isotype antibody (left), isotype-S239C-PBD-E2ADC (middle) and huM25-S239C-PBD-E2 (right), showing 80%, 90%, and 60%α-SMA tumor positivity, respectively. The α-SMA quantification acrosssamples is depicted in FIG. 24C, indicating a trend to lowering α-SMAupon treatment with huLRRC15-specific ADC. Additionally, FIG. 24D showsan increase in both F4/80 and CD11c expression with treatment ofhuLRRC15 ADC, suggesting an initiation of an immunological responsewithin the tumor.

FIG. 24E depicts another exemplary embodiment of anADC—huM25-S239C-PBD-E2—that comprised a DNA-damaging payload and led toanti-tumor activity in vivo in a NCI-H1650 xenograft model. NCI-H1650NSCLC cancer cells (5 million) were implanted subcutaneously intoSCID/Beige mice, and mice were randomized when the tumors reached ˜200mm³ and dosed on day 0 with isotype or ADC at 0.1, 0.3, or 0.6 mg/kgintraperitoneally. The ADC huM25-S239C-PBD-E2 showed a dose-dependentanti-tumor effect at 0.1, 0.3, and 0.6 mg/kg as compared with isotypeantibody, when measured by tumor volume in number of days post sizematchand a single dose of the ADC.

FIG. 24F shows that the LRRC15-targeting ADC huM25-S239C-PBD-E2 induceda statistically significant anti-tumor effect on tumor volume in aNCI-H1650 xenograft model (p<0.05), when compared to the isotype ADCwith the same DNA-damaging payload at the same concentration, consistentwith targeted delivery of the huLRRC15 antibody component causinglocalization of the ADC, thereby allowing the DNA-damaging payload toenhance cancer cell killing.

FIG. 24G depicts another exemplary embodiment of anADC—huAD208.4.1-PBD-DAR2—that comprised a DNA-damaging payload and ledto anti-tumor activity in vivo. NCI-H1650 adeno NSCLC cells (5 million)were implanted subcutaneously into SCID/Beige mice, and mice wererandomized when the tumors reached ˜225 mm³ and dosed with ADC at 0.6mg/kg or isotype antibody at 12 mg/kg intraperitoneally once on day 0.The anti-tumor efficacy for this anti-huLRRC15 PBD ADC(“huAD208.4.1-PBD-DAR2”) was TGI_(max) of 95%, and TGD of >388%.

In FIG. 24H, EBC-1 squamous NSCLC cells (5 million) were implantedsubcutaneously into SCID mice, and mice were randomized when the tumorsreached ˜225 mm³ and dosed with ADC at 0.6 mg/kg Q7D×2 (one dose givenevery 7 days for a total of 2 doses) or isotype antibody at 6 mg/kgintraperitoneally starting on day 0. The anti-tumor efficacy ofhuAD208.4.1-PBD-DAR2 was TGI_(max) of 91%, and TGD of >600%.

The potent anti-tumor activity shown in FIGS. 24A-24H with anti-huLRRC15ADCs comprising the DNA-damaging cytostatic and/or cytotoxic agent PBDdemonstrates that the huLRRC15 target may be used to deliveranti-huLRRC15 ADCs containing cytostatic and/or cytotoxic agents withdiffering mechanisms of action (e.g., DNA damage viapyrrolobenzodiazepine delivery) to the tumor site to elicit ananti-tumor response. These findings suggest different anti-huLRRC15antibodies (e.g., huAD208.4.1, huM25, huAD208.14.1, hu139.10,muAD210.40.9, or muAD208.9.1) can be conjugated with differingcytostatic and/or cytotoxic agents to successfully deliver the agent(s)to a tumor and induce cancer growth inhibition.

9. EMBODIMENTS

1. An antibody or binding fragment thereof that specifically bindshuLRRC15 extracellular domain, wherein said extracellular domaincomprises the proteolytic cleavage site defined by Arg⁵²⁷ and Ser⁵²⁸ ofSEQ ID NO:3.

2. The antibody or binding fragment of Embodiment 1 that competes forbinding cells expressing human LRRC15 with a control antibody selectedfrom huM25, huAD208.4.1, huAD208.12.1, huAD208.14.1, hu139.10,muAD210.40.9, and muAD209.9.1.

3. The antibody or binding fragment of Embodiment 1 in which the controlantibody is huM25.

4. The antibody or binding fragment of Embodiment 1 in which the controlantibody is huAD208.4.1.

5. The antibody or binding fragment of Embodiment 1, which comprises aV_(H) chain having three CDRs in which:

V_(H) CDR#1 corresponds in sequence to SEQ ID NO:10, V_(H) CDR#2corresponds in sequence to SEQ ID NO:11 and V_(H) CDR#3 corresponds insequence to SEQ ID NO:12;

V_(H) CDR#1 corresponds in sequence to SEQ ID NO:20, V_(H) CDR#2corresponds in sequence to SEQ ID NO:21 and V_(H) CDR#3 corresponds insequence to SEQ ID NO:22;

V_(H) CDR#1 corresponds in sequence to SEQ ID NO:30, V_(H) CDR#2corresponds in sequence to SEQ ID NO:31 and V_(H) CDR#3 corresponds insequence to SEQ ID NO:32;

V_(H) CDR#1 corresponds in sequence to SEQ ID NO:40, V_(H) CDR#2corresponds in sequence to SEQ ID NO:41 and V_(H) CDR#3 corresponds insequence to SEQ ID NO:42;

V_(H) CDR#1 corresponds in sequence to SEQ ID NO:50, V_(H) CDR#2corresponds in sequence to SEQ ID NO:51 and V_(H) CDR#3 corresponds insequence to SEQ ID NO:52;

V_(H) CDR#1 corresponds in sequence to SEQ ID NO:60, V_(H) CDR#2corresponds in sequence to SEQ ID NO:61 and V_(H) CDR#3 corresponds insequence to SEQ ID NO:62; or

V_(H) CDR#1 corresponds in sequence to SEQ ID NO:70, V_(H) CDR#2corresponds in sequence to SEQ ID NO:71 and V_(H) CDR#3 corresponds insequence to SEQ ID NO:72.

6. The antibody or binding fragment of Embodiment 1, which comprises aV_(L) chain having three CDRs in which:

V_(L) CDR#1 corresponds in sequence to SEQ ID NO:13, V_(L) CDR#2corresponds in sequence to SEQ ID NO:14 and V_(L) CDR#3 corresponds insequence to SEQ ID NO:15;

V_(L) CDR#1 corresponds in sequence to SEQ ID NO:23, V_(L) CDR#2corresponds in sequence to SEQ ID NO:24 and V_(L) CDR#3 corresponds insequence to SEQ ID NO:25;

V_(L) CDR#1 corresponds in sequence to SEQ ID NO:33, V_(L) CDR#2corresponds in sequence to SEQ ID NO:34 and V_(L) CDR#3 corresponds insequence to SEQ ID NO:35;

V_(L) CDR#1 corresponds in sequence to SEQ ID NO:43, V_(L) CDR#2corresponds in sequence to SEQ ID NO:44 and V_(L) CDR#3 corresponds insequence to SEQ ID NO:45;

V_(L) CDR#1 corresponds in sequence to SEQ ID NO:53, V_(L) CDR#2corresponds in sequence to SEQ ID NO:54 and V_(L) CDR#3 corresponds insequence to SEQ ID NO:55;

V_(L) CDR#1 corresponds in sequence to SEQ ID NO:63, V_(L) CDR#2corresponds in sequence to SEQ ID NO:64 and V_(L) CDR#3 corresponds insequence to SEQ ID NO:65; or

V_(L) CDR#1 corresponds in sequence to SEQ ID NO:73, V_(L) CDR#2corresponds in sequence to SEQ ID NO:74 and V_(L) CDR#3 corresponds insequence to SEQ ID NO:75.

7. The antibody or binding fragment of Embodiment 1 which comprises aV_(H) chain corresponding in sequence to SEQ ID NO:16 and a V_(L) chaincorresponding in sequence to SEQ ID NO:17.

8. The antibody or binding fragment of Embodiment 1 which comprises aV_(H) chain corresponding in sequence to SEQ ID NO:26 and a V_(L) chaincorresponding in sequence to SEQ ID NO:27.

9. The antibody or binding fragment of any one of Embodiments 1-7 whichcomprises a heavy chain corresponding in sequence to SEQ ID NO:18 and alight chain corresponding in sequence to SEQ ID NO:19.

10. The antibody of Embodiment 1 which is an IgG₁.

11. The antibody of Embodiment 1 having one or more reduced cysteineresidues bearing a free sulfhydryl group.

12. An antibody drug conjugate (“ADC”) comprising a cytotoxic and/orcytostatic agent linked to an antibody by way of a linker, wherein theantibody is an antibody according to any one of Embodiments 1-11, thecytotoxic and/or cytostatic agent is capable of traversing a cellmembrane, and the linker is cleavable by a lysosomal enzyme.13. The ADC of Embodiment 12 which has an average drug-to-antibody ratioin the range of 1-10.14. The ADC of Embodiment 12 which has an average drug-to-antibody ratioin the range of 2-4.15. The ADC of Embodiment 12 in which the lysosomal enzyme is CathepsinB.16. The ADC of Embodiment 15 in which the linker comprises a segmentaccording to structural formula (IVa), (IVb), (IVc), or (IVd):

or a salt thereof, wherein:

peptide represents a peptide (illustrated C→N and not showing thecarboxy and amino “termini”) cleavable by a lysosomal enzyme;

T represents a polymer comprising one or more ethylene glycol units oran alkylene chain, or combinations thereof;

R^(a) is selected from hydrogen, alkyl, sulfonate and methyl sulfonate;

p is an integer ranging from 0 to 5;

q is 0 or 1;

x is 0 or 1;

y is 0 or 1;

represents the point of attachment of the linker to a cytotoxic and/orcytostatic agent; and

* represents the point of attachment to the remainder of the linker.

17. The ADC of Embodiment 16 in which peptide is selected from the groupconsisting of Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit;Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys;Asp-Cit; Cit-Asp; Ala-Val; and Val-Ala and salts thereof.18. The ADC of Embodiment 12 in which the lysosomal enzyme isβ-glucuronidase.19. The ADC of Embodiment 12 in which the cytotoxic and/or cytostaticagent is MMAE.20. The ADC of Embodiment 12 in which the cytotoxic and/or cytostaticagent is a PBD dimer.21. The ADC of Embodiment 12 in which the antibody comprises three V_(H)CDRs corresponding in sequence, respectively, to SEQ ID NO:10, SEQ IDNO:11 and SEQ ID NO:12 and three V_(L) CDRs corresponding in sequence,respectively, to SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15.22. The ADC of Embodiment 21 in which the antibody comprises a V_(H)corresponding in sequence to SEQ ID NO:16.23. The ADC of Embodiment 21 in which the antibody comprises a V_(L)corresponding in sequence to SEQ ID NO:17.24. The ADC of Embodiment 21 in which the antibody comprises a V_(H)corresponding in sequence to SEQ ID NO:16 and a V_(L) corresponding toSEQ ID NO:17.25. The ADC of Embodiment 24 which is an IgG₁.26. The ADC of Embodiment 21 in which the antibody comprises three V_(H)CDRs corresponding in sequence, respectively, to SEQ ID NO:20, SEQ IDNO:21 and SEQ ID NO:22 and three V_(L) CDRs corresponding in sequence,respectively, to SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25.27. The ADC of Embodiment 26 in which the antibody comprises a V_(H)chain corresponding in sequence to SEQ ID NO:26.28. The ADC of Embodiment 26 in which the antibody comprises a V_(L)chain corresponding in sequence to SEQ ID NO:27.29. The ADC of Embodiment 26 in which the antibody comprises a V_(H)chain corresponding in sequence to SEQ ID NO:26 and a V_(L) chaincorresponding in sequence to SEQ ID NO:27.30. The ADC of Embodiment 29 which is an IgG₁.31. The ADC of Embodiment 12 in which the antibody is huM25.32. The ADC of Embodiment 12 in which the antibody is huAD208.4.1.33. The ADC of Embodiment 12 in which the antibody competes for bindinghuLRRC15 with huM25 in an in vitro assay.34. The ADC of Embodiment 12 in which the antibody competes for bindinghuLRRC15 with huAD208.4.1 in an in vitro assay.35. The ADC of Embodiment 12 which is a compound according to structuralformula (I):[D-L-XY-]_(n)-Ab  (I)

or a salt thereof, wherein:

D is the cytotoxic and/or cytostatic agent;

L is the linker;

Ab is the antibody;

XY represents a covalent linkage linking linker L to antibody Ab; and

n is an integer ranging from 2 to 8.

36. The ADC of Embodiment 35 in which n is 2, 3 or 4.

37. The ADC of Embodiment 35 in which XY is a linkage formed with anamino group on antibody Ab.

38. The ADC of Embodiment 37 in which XY is an amide or a thiourea.

39. The ADC of Embodiment 35 in which XY is a linkage formed with asulfydryl group on antibody Ab.

40. The ADC of Embodiment 39 in which XY is a thioether.

41. The ADC of Embodiment 35 in which the compound according tostructural formula (I) has the structure of formula (IIa):

where Ab is antibody huM25.42. The ADC of Embodiment 35 which has the structure:

where Ab is antibody huM25 and n is 2 or 4.43. The ADC of Embodiment 35 in which the compound according tostructural formula (I) has the structure of formula (IIb):

where Ab is antibody huAD208.4.1.44. The ADC of Embodiment 35 which has the structure:

where Ab is huAD208.4.1 and n is 2 or 4.45. A composition comprising an ADC according to any one of Embodiments12-44 and a carrier, excipient and/or diluent.46. The composition of Embodiment 45 which is formulated forpharmaceutical use in humans.47. The composition of Embodiment 45 which is in unit dosage form.48. An ADC formed by contacting an antibody that specifically bindshuLRRC15 extracellular domain, wherein said extracellular domaincomprises the proteolytic cleavage site defined by Arg⁵²⁷ and Ser⁵²⁸ ofSEQ ID NO:3, with a synthon according to structural formula (III) D-L-W,where D is a cytotoxic and/or cytostatic agent capable of crossing acell membrane, L is a linker cleavable by a lysosomal enzyme and R^(x)comprises a functional group capable of covalently linking the synthonto the antibody, under conditions in which the synthon covalently linksthe synthon to the antibody.49. The ADC of Embodiment 48 in which the antibody is huM25 and thecytotoxic and/or cytostatic agent is MMAE.50. The ADC of Embodiment 49 which has the structure

where Ab is the antibody, and n is 2 or 4.51. The ADC of Embodiment 48 in which the antibody is huAD208.4.1 andthe cytotoxic and/or cytostatic agent is a PBD dimer.52. The ADC of Embodiment 51 which has the structure

where Ab is the antibody, and n is 2 or 4.53. The ADC according to any one of Embodiments 48-52 in which thecontacting step is carried out under conditions such that the ADC has aDAR of 2, 3 or 4.54. A composition comprising an ADC according to Embodiment 48 and anexcipient, carrier and/or diluent.55. The composition of Embodiment 54 which is formulated forpharmaceutical use in humans.56. The composition of Embodiment 55 which is in unit dosage form.57. A method of making an ADC, comprising contacting an antibody thatspecifically binds huLRRC15 extracellular domain, wherein saidextracellular domain comprises the proteolytic cleavage site defined byArg⁵²⁷ and Ser⁵²⁸ of SEQ ID NO:3, with a synthon according to structuralformula (III) D-L-W, where D is cytotoxic and/or cytostatic agentcapable of crossing a cell membrane, L is a linker capable of beingcleaved by a lysosomal enzyme, and R^(x) comprises a functional groupcapable of covalently linking the synthon to the antibody, underconditions in which the synthon covalently links the synthon to theantibody.58. The method of Embodiment 57 in which the antibody is huM25 and thecytotoxic and/or cytostatic agent is MMAE.59. The method of Embodiment 57 in which the antibody is huAD208.4.1 andthe cytotoxic and/or cytostatic agent is a PBD dimer.60. A method of treating a huLRRC15 stromal(+)/cancer(−) tumor,comprising administering to a human having a huLRRC15stromal(+)/cancer(−) tumor an amount of an ADC according to any one ofEmbodiments 12-44 sufficient to provide therapeutic benefit.61. The method of Embodiment 60 in which the huLRRC15stromal(+)/cancer(−) tumor is relapsed, refractory, or relapsed andrefractory.62. The method of Embodiment 60 in which the huLRRC15stromal(+)/cancer(−) tumor is breast cancer, lung cancer, head and neckcancer, pancreatic cancer, colorectal cancer, ovarian cancer, testicularcancer, bladder cancer, or renal cancer.63. The method of Embodiment 60 in which the huLRRC15stromal(+)/cancer(−) tumor is a metastatic cancer.64. The method of Embodiment 60 in which the ADC is administered asmonotherapy.65. The method of Embodiment 64 in which the ADC is administeredintravenously at a dose ranging from about 0.3 mg/kg to about 6.0 mg/kg.66. The method of Embodiment 60 in which the ADC is administeredadjunctive to or with another anti-cancer therapy or agent.67. The method of Embodiment 66 in which the anti-cancer therapy oragent is a non-targeted anti-cancer therapy.68. The method of Embodiment 67 in which the non-targeted anti-cancertherapy is cisplatin, gemcitabine, docetaxel, carboplatin, or radiation.69. The method of Embodiment 66 in which anti-cancer therapy or agent isa targeted anti-cancer agent.70. The method of Embodiment 69 in which the huLRRC15stromal(+)/cancer(−) tumor is breast cancer and the targeted anti-canceragent is trastuzumab, pertuzumab, ado-trastuzumab emtansine,pembrolizumab, nivolumab, lapatinib, palbociclib, or everolimus.71. The method of Embodiment 69 in which the huLRRC15stromal(+)/cancer(−) tumor is NSC lung cancer and the targetedanti-cancer agent is bevacizumab, ramucirumab, pembrolizumab, nivolumab,atezolizumab, erlotinib, afatinib, gefitinib, crizotinib, or ceritinib.72. The method of Embodiment 69 in which the huLRRC15stromal(+)/cancer(−) tumor is head and neck cancer and the targetedanti-cancer agent is cetuximab, panitumumab, zalutumumab, nimotuzumab,pembrolizumab, nivolumab, gefinitib, erlotinib, lapatinib, afatinib,dacomitinib, bevacizumab, sorafenib, sunitinib, or vandetanib.73. The method of Embodiment 69 in which the huLRRC15stromal(+)/cancer(−) tumor is pancreatic cancer and the targetedanti-cancer agent is pembrolizumab, nivolumab, sunitinib, everolimus, orerlotinib.74. The method of Embodiment 69 in which the huLRRC15stromal(+)/cancer(−) tumor is colorectal cancer and the targetedanti-cancer agent is bevacizumab, ramucirumab, ziv-aflibercept,cetuximab, pembrolizumab, nivolumab, durvalumab, atezolizumab, orregorafenib.75. The method of Embodiment 69 in which the huLRRC15stromal(+)/cancer(−) tumor is ovarian cancer and the targetedanti-cancer agent is bevacizumab, pembrolizumab, nivolumab, aflibercept,nintedanib, trebananib, pazopanib, sunitinib, sorafenib, cediranib,olaparib, or niraparib.76. The method of Embodiment 60 in which the ADC is administeredadjunctively with or to a non-targeted anti-cancer therapy and atargeted anti-cancer agent.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. An anti-huLRRC15 antibody drug conjugate (ADC)comprising an antimitotic agent linked to an anti-huLRRC15 antibody byway of a linker, wherein the ADC has a structure of formula (I):[D-L-XY]_(n)-Ab  (I) or a salt thereof, wherein: D is the antimitoticagent; L is the linker; Ab is the anti-huLRRC15 antibody; XY representsa covalent linkage linking linker L to antibody Ab; and n is an integerranging from 2 to 8; wherein Ab comprises a V_(H) chain comprising thesequence of SEQ ID NO:16 and a V_(L) chain comprising the sequence ofSEQ ID NO:17, a V_(H) chain comprising the sequence of SEQ ID NO:26 anda V_(L) chain comprising the sequence of SEQ ID NO:27, a V_(H) chaincomprising the sequence of SEQ ID NO:36 and a V_(L) chain comprising thesequence of SEQ ID NO:37, a V_(H) chain comprising the sequence of SEQID NO:46 and a V_(L) chain comprising the sequence of SEQ ID NO:47, aV_(H) chain comprising the sequence of SEQ ID NO:56 and a V_(L) chaincomprising the sequence of SEQ ID NO:57, a V_(H) chain comprising thesequence of SEQ ID NO:66 and a V_(L) chain comprising the sequence ofSEQ ID NO:67, or a V_(H) chain comprising the sequence of SEQ ID NO:76and a V_(L) chain comprising the sequence of SEQ ID NO:77.
 2. The ADC ofclaim 1 in which n is 2, 3 or
 4. 3. The ADC of claim 1 in which XY is athioether linkage formed with a sulfydryl group on antibody Ab.
 4. TheADC of claim 1 in which L comprises Val-Cit or Val-Ala.
 5. The ADC ofclaim 1 in which D is an auristatin.
 6. The ADC of claim 1 in which Abis an IgG₁.
 7. The ADC of claim 1 which has a structure of formula(IIa):


8. The ADC of claim 7 in which n is 2, 3 or
 4. 9. The ADC of claim 7 inwhich Ab comprises: (a) a heavy chain having the amino acid sequence ofSEQ ID NOS: 18, 100, 102, or 103; and a light chain of SEQ ID NO:19; or(b) a heavy chain having the amino acid sequence of SEQ ID NOS: 28, 101,104, or 105; and a light chain of SEQ ID NO:29.
 10. The ADC of claim 7which has a structure of formula (IIIa):


11. The ADC of claim 10 in which n is 2 or
 4. 12. The ADC of claim 10 inwhich Ab comprises: (a) a heavy chain having the amino acid sequence ofSEQ ID NOS: 18, 100, 102, or 103; and a light chain of SEQ ID NO:19; or(b) a heavy chain having the amino acid sequence of SEQ ID NOS: 28, 101,104, or 105; and a light chain of SEQ ID NO:29.
 13. An anti-huLRRC15antibody drug conjugate (ADC) comprising a DNA intercalating agentlinked to an anti-huLRRC15 antibody by way of a linker, wherein the ADChas a structure of formula (I):[D-L-XY]_(n)-Ab  (I) or a salt thereof, wherein: D is the DNAintercalating agent; L is the linker; Ab is the anti-huLRRC15 antibody;XY represents a covalent linkage linking linker L to antibody Ab; and nis an integer ranging from 2 to 8; wherein Ab comprises a V_(H) chaincomprising the sequence of SEQ ID NO:16 and a V_(L) chain comprising thesequence of SEQ ID NO: 17, a V_(H) chain comprising the sequence of SEQID NO:26 and a V_(L) chain comprising the sequence of SEQ ID NO:27, aV_(H) chain comprising the sequence of SEQ ID NO:36 and a V_(L) chaincomprising the sequence of SEQ ID NO:37, a V_(H) chain comprising thesequence of SEQ ID NO:46 and a V_(L) chain comprising the sequence ofSEQ ID NO:47, a V_(H) chain comprising the sequence of SEQ ID NO:56 anda V_(L) chain comprising the sequence of SEQ ID NO:57, a V_(H) chaincomprising the sequence of SEQ ID NO:66 and a V_(L) chain comprising thesequence a SEQ ID NO:67, or a V_(H) chain comprising the sequence of SEQID NO:76 and a V_(L) chain comprising the sequence of SEQ ID NO:77. 14.The ADC of claim 13 in which n is 2, 3 or
 4. 15. The ADC of claim 13 inwhich XY is a thioether linkage formed with a sulfydryl group onantibody Ab.
 16. The ADC of claim 13 in which L comprises Val-Cit orVal-Ala.
 17. The ADC of claim 13 in which D is a pyrrolobenzodiazepine.18. The ADC of claim 13 in which Ab is an IgG₁.
 19. The ADC of claim 13which has a structure of formula (IIb):


20. The ADC of claim 19 in which n is 2, 3 or
 4. 21. The ADC of claim 19in which Ab comprises: (a) a heavy chain having the amino acid sequenceof SEQ ID NO: 18, 100, 102, or 103; and a light chain of SEQ ID NO:19;or (b) a heavy chain having the amino acid sequence of SEQ ID NO: 28,101, 104, or 105; and a light chain of SEQ ID NO:29.
 22. The ADC ofclaim 19 which has a structure of formula (Mb)


23. The ADC of claim 22 in which n is 2 or
 4. 24. The ADC of claim 22 inwhich Ab comprises: (a) a heavy chain having the amino acid sequence ofSEQ ID NOS: 18, 100, 102, or 103; and a light chain of SEQ ID NO:19; or(b) a heavy chain having the amino acid sequence of SEQ ID NOS: 28, 101,104, or 105; and a light chain of SEQ ID NO:29.
 25. A pharmaceuticalcomposition comprising an ADC of claim 1 and a pharmaceuticallyacceptable carrier.
 26. A pharmaceutical composition comprising an ADCof claim 13 and a pharmaceutically acceptable carrier.
 27. Ananti-huLRRC15 antibody or an anti-huLRRC15 binding fragment thereofwhich comprises: a V_(H) chain comprising the sequence of SEQ ID NO:16and a V_(L) chain comprising the sequence of SEQ ID NO:17; a V_(H) chaincomprising the sequence of SEQ ID NO:26 and a V_(L) chain comprising thesequence of SEQ ID NO:27; a V_(H) chain comprising the sequence of SEQID NO:36 and a V_(L) chain comprising the sequence of SEQ ID NO:37; aV_(H) chain comprising the sequence of SEQ ID NO:46 and a V_(L) chaincomprising the sequence of SEQ ID NO:47; a V_(H) chain comprising thesequence of SEQ ID NO:56 and a V_(L) chain comprising the sequence ofSEQ ID NO:57; a V_(H) chain comprising the sequence of SEQ ID NO:66 anda V_(L) chain comprising the sequence of SEQ ID NO:67; or a V_(H) chaincomprising the sequence of SEQ ID NO:76 and a V_(L) chain comprising thesequence of SEQ ID NO:77.